CN116685327A - Orphan nuclear receptor modulators for the treatment of pancreatitis, glioblastoma, sarcopenia and stroke - Google Patents

Abstract

Compounds, compositions, and methods for modulating retinoic acid receptor-like orphan receptors (RORs) to increase FGF21 levels, and to treat and prevent FGF 21-associated disorders, such as pancreatitis, sarcopenia, stroke, and traumatic brain injury, and to increase miR-122 levels, and to treat and prevent disorders, such as glioblastoma.

Description

Orphan nuclear receptor modulators for the treatment of pancreatitis, glioblastoma, sarcopenia and stroke

Cross Reference to Related Applications

The present application claims the priority under PCT 8 of U.S. provisional patent application No. 63/108,054 entitled "orphan nuclear receptor modulator for the treatment of pancreatitis, glioblastoma, sarcopenia, stroke, and traumatic brain injury" filed on 10/30 of 2020. The contents of the above-mentioned applications are incorporated herein by reference as if fully set forth herein in their entirety.

Technical Field

The present application relates to small molecule modulators of retinoic acid receptor-Related Orphan Receptors (RORs), such as rorα, rorβ or rorγ, for use in the treatment of conditions associated with FGF21 and/or miR122, such as pancreatitis, sarcopenia, stroke, glioblastoma, and traumatic brain injury.

Background

Pancreatitis is one of the most common and debilitating diseases of the gastrointestinal tract, leading to considerable morbidity and mortality. Pancreatitis is caused by the premature activation of digestive enzymes of the pancreas itself, which can lead to tissue damage and inflammation. Common causes of pancreatitis include alcoholism and gallstones. About one-third of human pancreatitis cases are caused by alcohol, with the highest incidence. Pancreatitis also occurs in 5-10% of patients undergoing Endoscopic Retrograde Cholangiopancreatography (ERCP), a procedure used to examine the pancreas and bile duct and the gallbladder.

There is no specific therapy for this severe clinical disease. Treatment of pancreatitis is often supportive. Thus, new therapies are urgently needed.

Pancreatitis begins with the activation of digestive enzymes in the pancreas, which results in tissue damage and inflammation. Common causes of pancreatitis include alcoholism, hyperlipidemia, and gall stones moving out of the biliary tract. Pancreatitis is also iatrogenic and occurs in 5-10% of patients receiving Endoscopic Retrograde Cholangiopancreatography (ERCP). Overall, pancreatitis is an unmet therapeutic need.

Pancreatitis is a condition of deficiency of fibroblast growth factor 21 (FGF 21) that can be corrected by increasing FGF21 levels. The relationship between pancreatitis and FGF21 is discussed below.

Fibroblast growth factor 21 (FGF 21) is a hormone secreted by the liver in response to various metabolic stresses including starvation and consumption of alcohol or monosaccharides. FGF21 acts on a heteromeric cell surface receptor complex consisting of a classical FGF receptor FGFR1c and a proprietary-co-receptor β -klotho (7-9). FGF21 is also highly expressed in the exocrine pancreas where it acts directly on acinar cells in an autocrine/paracrine fashion to stimulate secretion of digestive enzymes. This can prevent protein overload and relieve Endoplasmic Reticulum (ER) stress. Mice lacking FGF21 are particularly susceptible to sky blue pigment induced pancreatitis, an analog of cholecystokinin (CCK). In contrast, gene overexpression of FGF21 provided protection in this model. Likewise, prophylactic administration of FGF21 reduced fibrosis in a mouse model of l-arginine-induced chronic pancreatitis.

Exocrine pancreas expresses FGF21 at the highest concentration in vivo, thereby maintaining acinar cell protein homeostasis. As shown in the mouse and human models, acute and chronic pancreatitis is associated with the loss of FGF21 expression due to activation of the Integrated Stress Response (ISR) pathway. Mechanistically, activation of ISR in cultured acinar cells and mouse pancreas induces expression of ATF3 (a transcription inhibitor) to bind directly to specific sites on the Fgf21 promoter, resulting in loss of Fgf21 expression. These ATF3 binding sites are conserved in the human FGF21 promoter.

Consistent with the mouse study, mutual expression of ATF3 and FGF21 was observed in the pancreas of human pancreatitis patients. Drug replacement of FGF21 reduced ISR and resolved pancreatitis. Likewise, inhibition of ISR with PKR-like endoplasmic reticulum kinase (PERK) inhibitors also restored FGF21 expression and reduced pancreatitis. These findings underscore the importance of FGF21 in protecting pancreatic exocrine function.

Serum FGF21 levels are positively correlated with sarcopenia (see Tezze et al, age-associated loss of OPA1 in muscle impacts muscle mass, metabolic homeostasis, systemic inflammation, and Epithelial Senescience. Cell Metab 2017;25:1374-1389e 6).

FGF21 also has the effect of treating traumatic brain injury and Stroke (see, e.g., jiang et al, "Abstract WMP81: FGF21 reduction Post-Stroke Blood Brain Barrier Damage in Diabetic db/db Male Mice," Stroke, vol 51, issue support_1 (month 2 2020)). Jiang discloses that recombinant human fibroblast growth factor 21 (rFGF 21) protects BBB injury after stroke by pparγ activation of brain microvascular endothelium. See also Chen et al, "FGF21 Protects the Blood-Brain Barrier by Upregulating PPAR. Gamma. Via FGFR 1/beta. -klotho after Traumatic Brain Injury," Journal of Neurotrauma, vol.35, no.17 (2018)).

Blood Brain Barrier (BBB) disruption and dysfunction leads to cerebral oedema, which is responsible for more than half of the deaths following severe Traumatic Brain Injury (TBI). Chen discloses that fibroblast growth factor 21 (FGF 21) has a potential neuroprotective function in the brain. The effect of recombinant human FGF21 (rhFGF 21) on BBB integrity and Tight Junction (TJ) and Adhesion Junction (AJ) proteins was studied in a TBI mouse model and an in vitro BBB disruption model established with tumor necrosis factor alpha (TNF-alpha) induced Human Brain Microvascular Endothelial Cells (HBMECs). The ability of rhFGF21 to form FGF21/FGFR 1/beta-klotho complex was demonstrated by in vitro transfection of beta-klotho small interfering RNA (siRNA) and co-immunoprecipitation of FGFR 1. In a mouse model following TBI, rhFGF21 significantly reduced the extent of neurological behavioural deficits and cerebral edema, maintained the integrity of the BBB, and reduced brain tissue loss and neuronal apoptosis. In vivo and in vitro, rhFGF21 upregulates TJ and AJ proteins, thereby protecting the BBB. In addition, rhFGF21 activates PPARgamma in TNF-alpha induced HBMECs by forming FGF21/FGFR 1/beta-klotho complexes. rhFGF21 up-regulates the TJ and AJ proteins by FGF21/FGFR 1/beta-klotho complex formation and PPARgamma activation, thereby protecting the BBB. FGF21 is therefore useful for the treatment of traumatic brain injury and other diseases caused by BBB destruction, brain abscesses, in vitro diseases, HIV encephalitis, meningitis, multiple sclerosis, and neuromyelitis optica.

FGF21 is administered by injection and therefore for reasons of patient compliance it would be advantageous to provide compounds that can be administered orally to treat or prevent pancreatitis, sarcopenia, stroke, glioblastoma, or traumatic brain injury, or to reduce the susceptibility to, reduce the severity of, or delay the progression of such disorders. The present invention provides such compounds, and methods of using the same.

Disclosure of Invention

In one embodiment, rora agonist compounds, compositions comprising these compounds, and methods for treating or preventing pancreatitis, sarcopenia, stroke, glioblastoma, traumatic brain injury, or reducing the susceptibility to, reducing the severity of, or slowing the progression of these disorders are disclosed. In other embodiments, the compounds are useful for other disorders associated with FGF21 deficiency, or other disorders that may benefit from higher than normal FGF21 levels and/or miR 122.

Fibroblast growth factor 21 (FGF 21) is a hormone secreted by the liver in response to various metabolic stresses. FGF21 is expressed in the exocrine pancreas to stimulate digestive enzyme secretion. FGF21 Knockout (KO) mice are particularly prone to pancreatitis. Overexpression of FGF21 provides protection against pancreatitis. Prophylactic administration of FGF21 reduced fibrosis in a mouse model of pancreatitis. Loss of FGF21 is a driving factor for pancreatitis. FGF21 was used to therapeutically reverse pre-existing pancreatitis.

Because the rora agonists described herein increase expression of endogenous FGF21, rora agonists are useful for treating, preventing, reducing the susceptibility of, lessening the severity of, or slowing the progression of pancreatitis.

In some aspects of this embodiment, methods are provided for modulating ROR biological activity in a subject in a manner that increases the level of endogenous FGF21 in the subject. Increasing FGF21 levels treats, prevents, reduces the susceptibility to, reduces the severity of, or delays the progression of FGF 21-deficiency-related disorders, such as pancreatitis or sarcopenia, and also provides neuroprotection to help patients suffering from stroke, traumatic brain injury, and the like.

In other aspects of this embodiment, methods of modulating ROR biological activity in a subject in a manner that increases the level of endogenous miR122 in the subject are provided. Increasing miR122 levels treats, prevents, reduces the susceptibility to, reduces the severity of, or delays progression of miR 122-related disorders, such as those involving lipid droplet formation, such as glioblastoma and the like.

The method comprises contacting ROR with an effective amount of a compound of formula (a) as shown below, wherein the compound is an agonist or activator of RORA (also referred to herein as RORA).

In another embodiment, the compound has the general formula:

and pharmaceutically acceptable salts and prodrugs thereof, wherein R ² And u is as defined elsewhere herein for formula A.

A representative compound has the general formula:

and pharmaceutically acceptable salts and prodrugs thereof, wherein R ² And u is as defined elsewhere herein for formula A.

In another embodiment, the compound is benzodiazepine, which binds to the RORA receptor with relatively high affinity, is an agonist of the RORA receptor, and does not cross the blood brain barrier and/or does not bind to GABA receptors (e.g., GABA-a receptor) with high affinity. In this embodiment, the compounds are useful for treating a variety of conditions, including pancreatitis and sarcopenia associated with FGF 21.

In another embodiment, the compound is benzodiazepine, which binds to the RORA receptor with relatively high affinity, is an agonist of the RORA receptor, and does cross the blood brain barrier, but does not bind to GABA receptors (e.g., GABA-a receptor) with high affinity. In this embodiment, the compounds are useful for treating a variety of FGF 21-associated neurological disorders, including stroke and traumatic brain injury.

In some embodiments, benzodiazepine substitutions are used to treat fatty liver disease, such as NASH, and cirrhosis caused by fatty liver disease progression.

The compounds described herein may be in the form of stereoisomers, polymorphs, salts and prodrugs.

In various embodiments, pharmaceutical compositions and formulations are provided comprising an effective compound of formulas (a) - (H) to treat, prevent, reduce the susceptibility to, reduce the severity of, or delay the progression of conditions associated with FGF21 deficiency, such as pancreatitis, sarcopenia, stroke, and traumatic brain injury. The composition may comprise a compound of formulae (a) - (H) and a pharmaceutically acceptable carrier or excipient, and may optionally comprise one or more additional active agents.

In various embodiments, pharmaceutical compositions and formulations are provided comprising an effective compound of formulas (B) - (H) to treat, prevent, reduce the susceptibility to, reduce the severity of, or delay the progression of conditions associated with FGF21 deficiency, such as pancreatitis, sarcopenia, stroke, and traumatic brain injury. The composition may comprise a compound of formulae (B) - (H) and a pharmaceutically acceptable carrier or excipient, and may optionally comprise one or more additional active agents. R of the formula A is specifically listed ¹ The variables may also be used in any of formulas (B) - (H).

The invention will be better understood with reference to the following detailed description.

Drawings

FIG. 1 is a graph showing how Compound 1 induces RORα -regulated luciferase expression with WT RORE, but does not have activity when RORE is mutated. Data are expressed as relative luciferase activity versus concentration, error line = SD. P <0.05 compared to DMSO.

FIG. 2 is a graph showing how compound 1 specifically increases miR-122 secretion from Huh-7 cells, expressed as relative DMSO control and relative microRNA levels of compound 1 (1. Mu.M) in Huh7 medium. Data are represented by error line = SD. * P <0.01.

Figures 3A-B are graphs showing how compound 1 modulates Th17 populations in human peripheral mononuclear cells (PBMCs). FIG. 3A shows how the viability of CD4+ T cells is determined by LIVE/DEAD fixable water-soluble DEAD cell staining, expressed as% viability in the entire CD4+ Th17 cell population. FIG. 3B shows the total percent composition of CD4+ Th17 (expressed as% Th17 cells) as determined by gating on CD3+/CD4+/CD45RA-/CXCR3-/CCR4+ CXCR5-/CCR6+ cells. These results indicate that compound 1 selectively reduced the cd4+ Th17 population under stimulated conditions.

Fig. 4A-E are graphs showing how compound 1 increases rorα target gene expression-miR-122 and Gpase6 in mice (n=3). Fig. 4A shows plasma levels of miR-122 levels measured over 7 days, while fig. 4B shows liver levels thereof. FIG. 4C shows the measurement of mRNA levels of miR-122 and ROR alpha target genes (Aldria and Gpase6, respectively) and miR-122 precursors over 7 days. The data show that secreted miR-122 enters the surrounding tissue. Figures 4D and 4E show miR-122 levels in skeletal muscle (4D) and white adipose tissue ("WAT") (4E) measured within 7 days. The data shows that miR-18 and miR-126 are unaffected after treatment with compound 1. Data are represented by error line = SD. P <0.05, P <0.01, P <0.001 compared to normal saline. White bars are control (normal saline), red bars are results on day 1, pink bars are results on day 3, and purple bars are results on day 7.

FIGS. 5A-C are graphs showing that compound 1 (Cmpd 1) treatment reduced body weight and increased energy expenditure by miR-l22 activity in high-fat fed C57BL/6 mice. Figure 5A shows the change in body weight (g) before (blue) and after (red) 3 weeks of treatment. FIG. 5B shows qRT-PCR analysis of plasma relative miR-l22 levels at the final time point. Fig. 5C is a graph showing colorimetric quantification of β -hydroxybutyrate plasma levels (in nM) after 3 weeks of treatment. Fig. 5D is a photograph of representative lipid accumulation visualized by H & E staining of liver sections. N=5. Data are represented by error line = SD. * P <0.05, < P <0.01, < P <0.001.

Fig. 6A-B are graphs showing that administration of compound 1 increases miR-122 expression and decreases liver and muscle triglyceride levels in Sgp, 130FC mice (n=3). Sgp130FC mice were injected (ip) with compound 1 four weeks (7.5 mg/kg twice weekly). FIGS. 6A-6B show qRT-PCR analysis of miR-122 levels in plasma (6A) and liver (6B) after treatment with saline (as a control) and compound 1 for 4 weeks. MicroRNA-18 was used as a negative control, and its plasma and liver levels were unaffected after treatment with Compound 1. The effect seen on this microRNA was not significant in figure 6A compared to the significant effect seen on miR-122.

FIGS. 7A and 7B show the results of qRT-PCR analysis of miR-122 extracted from plasma and liver in mice treated with Compound 1 or physiological saline, respectively. FIG. 7C shows qRT-PCR analysis of FGF21 and G6pc extracted from mouse liver and ROR alpha target genes pri-miR-122 and pre-miR-122 mRNA. FIG. 7D is a graph showing quantification of liver Triglyceride (TG) levels (mg/dL) in mice administered with physiological saline or compound 1.

Figures 8A-D are graphs showing various markers of liver injury in C57BL/6 mice fed an atherogenic diet for 3 weeks (inducing fibrosis) and 3 times weekly for 3.5 weeks (n=8) with 15mg/kg of compound 1 (or physiological saline+dmso) injected. qRT-PCR analysis of miR-122 extracted from plasma (FIG. 8A) and liver (FIG. 8B) was used for untreated (grey bars) and treated (black bars) groupings. miR-93 and miR-18 were used as negative controls in plasma and liver, respectively. Fig. 8C is a graph showing ALT and AST plasma levels measured at the end of the experiment. FIG. 8D is a graph showing qRT-PCR analysis of mRNA of the gene involved in fibrosis and the ROR alpha target gene (FGF 21) extracted from mouse liver. Normalizing microRNA levels in plasma to caenorhabditis elegans (C.elegans) miR-39; microRNA levels in tissues were normalized to RNU6.mRNA levels were normalized to HPRT. Data are represented by error line = SD. * P <.05, P <.01.* P < 001, P <0.0001.

FIG. 9A shows representative photomicrographs of H & E, CD3 and F4/80 stained livers taken from normal saline or compound 1 treated mice; the scale bar represents 10 μm. FIG. 9B is a graph showing quantification of positively stained F4/80 regions using ImageJ.

FIGS. 10A and 10C are photomicrographs of Masson trichromatic (M.T.) stained and alpha-SMA stained livers taken from saline or compound 1 treated mice; the scale bar represents 10 μm. Fig. 10B and D are graphs showing quantification of positively stained areas using ImageJ (%). The m.t. staining is shown in fig. 10B, while the SMA staining is shown in fig. 10D.

FIG. 11 shows the expression levels of ROR alpha, ROR alpha and MIR122 target genes in the liver of NASH patients. Database-based gene expression analysis was performed using the human public dataset obtained from the NCBI GEO database (http:// www.ncbi.nlm.nih.gov/GEO /), processed to measure median normalized signal intensity values for liver mRNA levels in normal and steatohepatitis patients. In the liver of NASH patients, pre-MIR122 and rorα target gene (FGF 21) expression levels are positively correlated. The positive correlation coefficient (r 2) is calculated by Pearson correlation test. GSE89632, n=25 (normal) and N=14 (NASH) for GEO.

Figures 12-13 show that rora agonist compound 1 reduces steatosis by increasing MIR122 expression in HFD-fed mice. The figure shows the levels of MIR122 (Agpat 1 and Dgat 1) and ROR alpha (FGF 21) target genes in the liver and muscle. FIG. 13 shows RNA-seq analysis of RNA extracted from liver tissue, showing a positive correlation between pri-MIR122 and FGF21 (RORα target gene) mRNA expression. microRNA levels in plasma were normalized to caenorhabditis elegans spinosa miR-39; microRNA levels in tissues were normalized to RNU6.mRNA levels were normalized to HPRT. Data are expressed as mean ± s.d. N=6. * P <0.05, P <0.01.* P <0.001, P <0.0001.

FIG. 14 shows the results of qRT-PCR analysis of ROR alpha target gene and pri-MIR122 and pre-MIR122mRNA extracted from mouse liver. The data shows that rora agonist compound 1 increases the level of MIR 122.

Figure 15 shows the anti-inflammatory and anti-fibrogenic effects of compound 1. qRT-PCR analysis of mRNA of gene involved in fibrosis and ROR alpha target gene (FGF 21) extracted from mouse liver, wherein microRNA level in plasma is normalized to caenorhabditis elegans spinosa miR-39 and microRNA level in tissue is normalized to RNU6.mRNA levels were normalized to HPRT. Data are expressed as mean ± s.d. * P <0.05, P <0.01.* P <0.001.

FIG. 16 is a graph showing relative FGF21 expression based on different concentrations of SR1078 (μM).

FIG. 17 is a schematic representation of a ligand binding domain of a RORA receptor upon which a compound rests.

FIGS. 18A and B are schematic diagrams of putative agonists of RORA receptors binding to miR-122 promoters, showing how luciferase activity can be measured when an agonist binds to a receptor, and no luciferase activity when the compound is not an agonist.

FIG. 19 is a graph showing the relative luciferase activity of compound 68 at different concentrations for wild-type and mutant RORα.

Detailed Description

The compounds of formulas (a) - (H) described herein modulate the expression of ROR target genes in hepatocytes, particularly hepatocytes associated with miR-122 production and subsequent FGF21 production.

The increase in FGF21 production can be used to treat a variety of conditions including pancreatitis, sarcopenia, stroke, and traumatic brain injury associated with FGF 21.

Increased production of miR-122 also reduced lipid droplet formation, as a means to avoid lipotoxicity, glioblastoma (GBM) cells formed lipid droplets. Thus, administration of a compound described herein to a subject increases the endogenous miR122 level in the subject, thereby treating, preventing, reducing the susceptibility to, reducing the severity of, or slowing the progression of a miR-122-related disorder, such as a disorder involving lipid droplet formation, such as Glioblastoma (GBM), and the like.

Also disclosed are pharmaceutical formulations comprising one or more of the compounds described herein, and a pharmaceutically acceptable carrier or excipient. In one embodiment, the formulation comprises at least one compound described herein and at least one further therapeutic agent.

The invention will be better understood with reference to the following definitions.

I. Definition of the definition

The term "independently" is used herein to mean that the variables that are independently applied vary independently from application to application. Thus, in a compound such as R "XY", where R "is independently carbon or nitrogen, then both R" may be carbon, both R "may be nitrogen, or one R" may be carbon and the other R "nitrogen.

The term "modulator" includes antagonists, allosteric inhibitors, agonists and partial agonists. Certain modulators may shut down ROR expression (direct antagonists and allosteric inhibitors, as well as partial agonists in a dose-dependent manner), while other modulators (agonists and partial agonists, the latter in a dose-dependent manner) may increase ROR expression.

As used herein, the term "enantiomerically pure" refers to a compound composition comprising at least about 95%, preferably about 97%, 98%, 99% or 100% of a single enantiomer of the compound.

As used herein, the term "substantially free" or "substantially absent" refers to a compound composition comprising at least 85% to 90% by weight, preferably 95% to 98% by weight, even more preferably 99% to 100% by weight of the specified enantiomer of the compound. In a preferred embodiment, the compounds described herein are substantially free of enantiomers.

Similarly, the term "isolated" refers to a compound composition comprising at least 85% to 90% by weight, preferably 95% to 98% by weight, even more preferably 99% to 100% by weight of the compound, the remainder comprising other chemicals or enantiomers.

The term "alkyl" as used herein, unless otherwise indicated, refers to saturated straight, branched or cyclic, primary, secondary or tertiary hydrocarbon compounds, including substituted and unsubstituted alkyl groups. Alkyl groups may optionally be substituted with any moiety that does not otherwise interfere with the reaction or provide improvement in the process, including but not limited to halogen, haloalkyl, hydroxy, carboxy, acyl, aryl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, thiol, imine, sulfonyl, sulfanyl, sulfinyl, sulfamoyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether, acyl halide, anhydride, oxime, hydrazine, carbamate, phosphonic acid, phosphonate, which are unprotected or optionally protected as known to those skilled in the art, e.g., as Greene et al, Protective Groups in Organic SynthesisJohn Wiley and Sons, second edition, 1991, incorporated herein by reference. Specifically include CF ₃ And CH (CH) ₂ CF ₃ . When an alkyl moiety is substituted at both termini, it is an "alkylene" moiety, such as a methylene moiety, and is intended to be included herein.

In this document, whenever the term C (alkyl range) is used, the term includes each member of the class independently as if specifically and individually listed. The term "alkyl" includes C _1-22 Alkyl moieties, the term "lower alkyl" includes C _1-6 An alkyl moiety. Those of ordinary skill in the art will appreciate that the relevant alkyl groups are named by replacing the suffix "-alkane (-ane)" with the suffix "-yl)".

As used herein, "bridged alkyl" refers to a bicyclo or tricyclic hydrocarbon, e.g., 2:1:1 bicyclohexane.

As used herein, "spiroalkyl" refers to two rings attached to a single (quaternary) carbon atom.

The term "alkenyl" refers to an unsaturated hydrocarbon radical, straight or branched, so long as it contains one or more double bonds. The alkenyl groups disclosed herein may be optionally substituted with any moiety that does not adversely affect the reaction process, including but not limited to those described for substituents on the alkyl moiety. Non-limiting examples of alkenyl groups include ethylene, methyl ethylene, isopropylidene, 1, 2-ethane-diyl, 1-ethane-diyl, 1, 3-propane-diyl, 1, 2-propane-diyl, 1, 3-butane-diyl and 1, 4-butane-diyl.

The term "alkynyl" refers to an unsaturated acyclic hydrocarbon radical, straight or branched, so long as it contains one or more triple bonds. The alkynyl group may be optionally substituted with any moiety that does not adversely affect the reaction process, including but not limited to those described above for the alkyl moiety. Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, hydroxypropionyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl, pentyn-2-yl, 4-methoxypentan-2-yl, 3-methylbutyn-1-yl, hexyn-2-yl and hexyn-3-yl, 3-dimethylbutyyn-1-yl.

The term "alkylamino" or "arylamino" refers to an amino group having one or two alkyl or aryl substituents, respectively.

As used herein, the term "fatty alcohol" refers to a linear primary alcohol having from 4 to 26 carbons in the chain, preferably from 8 to 26 carbons in the chain, and most preferably from 10 to 22 carbons in the chain. The exact chain length varies with the source. Representative fatty alcohols include lauryl alcohol, stearyl alcohol, and oleyl alcohol. They are colorless oily liquids (for smaller carbon numbers) or waxy solids, but impure samples may exhibit a yellow color. Fatty alcohols typically have an even number of carbon atoms and an alcohol group (-OH) attached to the terminal carbon. Some are unsaturated and some are branched. They are widely used in industry. As with fatty acids, they are generally represented by the number of carbon atoms in the molecule, e.g. "C ₁₂ Alcohols ", i.e. alcohols having 12 carbons, such as dodecanol.

The term "protected" as used herein, unless otherwise defined, refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent further reaction or for other purposes. A variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis and are described, for example, in Greene et al, protective Groups in Organic Synthesis, supra.

The term "aryl" used alone or in combination refers to a carbocyclic aromatic system containing one, two, or three rings, wherein the rings may be linked together in a pendent manner or may be fused. Non-limiting examples of aryl groups include phenyl, biphenyl, or naphthyl, or other aromatic groups that remain after hydrogen is removed from the aromatic ring. The term aryl includes both substituted and unsubstituted moieties. The aryl group may be optionally substituted with any moiety that does not adversely affect the process, including but not limited to those described above for the alkyl moiety. Non-limiting examples of substituted aryl groups include heteroarylamino, N-aryl-N-alkylamino, N-heteroarylamino-N-alkylamino, heteroarylalkoxy, arylamino, aralkylamino, arylthio, monoarylamidosulfonyl, arylsulfinylamino, diarylcamidosulfonyl, monoarylamidosulfonyl, arylsulfinyl, heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, aroyl, heteroarylacyl, aralkanoyl, heteroarylalkanoyl, hydroxyarylalkyl, hydroxyheteroarylalkyl, haloalkoxyalkyl, aryl, aralkyl, aryloxy, aralkoxy (arylkony), aryloxyalkyl, saturated heterocyclyl, partially saturated heterocyclyl, heteroaryl, heteroaryloxy, heteroaryloxyalkyl, aralkyl, heteroarylalkyl, arylalkenyl, and heteroarylalkenyl groups.

The term "alkylaryl" or "alkylaryl" refers to an alkyl group having an aryl substituent. The term "aralkyl" or "arylalkyl" refers to an aryl group having an alkyl substituent.

The term "halogen" as used herein includes chlorine, bromine, iodine and fluorine.

The term "acyl" refers to a carboxylic acid ester wherein the non-carbonyl moiety of the ester group is selected from the group consisting of: straight, branched or cyclic alkyl or lower alkyl, alkoxyalkyl (including but not limited to methoxymethyl), aralkyl (including but not limited to benzyl), aryloxyalkyl (such as phenoxymethyl), aryl (including but not limited to phenyl), optionally substituted with: halogen (F, cl, br or I), alkyl (including but not limited to C ₁ 、C ₂ 、C ₃ And C ₄ ) Or alkoxy (including but not limited toIn C ₁ 、C ₂ 、C ₃ And C ₄ ) Sulfonate esters (such as alkyl or aralkylsulfonyl groups including, but not limited to, methylsulfonyl), mono-, di-or triphosphate esters, trityl or monomethoxytrityl, substituted benzyl, trialkylsilyl (e.g., dimethyl-t-butylsilyl) or diphenylmethylsilyl. The aryl groups in the esters optimally comprise phenyl groups. The term "lower acyl" refers to an acyl group wherein the non-carbonyl moiety is lower alkyl.

The terms "alkoxy" and "alkoxyalkyl" include straight or branched chain oxygen containing groups having an alkyl moiety, such as methoxy. The term "alkoxyalkyl" also includes alkyl groups having one or more alkoxy groups attached to the alkyl group, i.e., forming monoalkoxyalkyl and dialkoxyalkyl groups. The "alkoxy" group may be further substituted with one or more halogen atoms, such as fluorine, chlorine or bromine, to provide a "haloalkoxy" group. Examples of such groups include fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy, trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy and fluoropropoxy.

The term "alkylamino" means "mono-alkylamino" and "dialkylamino" containing one or two alkyl groups, respectively, attached to an amino group. The term arylamino denotes "monoarylamino" and "diarylamino" groups containing one or two aryl groups, respectively, attached to an amino group. The term "aralkylamino" includes aralkyl groups attached to an amino group. The term aralkylamino refers to "monoarylamino" and "diarylamino" containing one or two aralkyl groups, respectively, attached to an amino group. The term aralkylamino also denotes a "monoarylalkylmonoalkylamino" group containing one aralkyl group and one alkyl group attached to an amino group.

The term "heteroatom" as used herein refers to oxygen, sulfur, nitrogen and phosphorus.

The term "heteroaryl" or "heteroaromatic" as used herein refers to an aromatic compound comprising at least one sulfur, oxygen, nitrogen, or phosphorus in an aromatic ring.

The terms "heterocycle", "heterocyclyl" and cycloheteroalkyl refer to a non-aromatic cyclic group in which at least one heteroatom, such as oxygen, sulfur, nitrogen or phosphorus, is present in the ring.

Non-limiting examples of heteroaryl and heterocyclic groups include furyl (furyl), pyridyl, pyrimidinyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuryl, benzothienyl, quinolinyl, isoquinolinyl, benzothienyl, isobenzofuranyl, pyrazolyl, indolyl, isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2, 4-thiadiazolyl, isoxazolyl, pyrrolyl, quinazolinyl, cinnolinyl, phthalazinyl, xanthinyl, hypoxanthyl (hypoxanyl), thiophene, furan, pyrrole isopyrroles, pyrazoles, imidazoles, 1,2, 3-triazoles, 1,2, 4-triazoles, oxazoles, isoxazoles, thiazoles, isothiazoles, pyrimidines or pyridazines and pteridines, aziridines, thiazoles, isothiazoles, 1,2, 3-oxadiazoles, thiazines, pyridines, pyrazines, piperazines, pyrrolidines, oxaziranes, phenazines, phenothiazines, morpholines, pyrazoles, pyridazines, pyrazines, quinoxalines, xanthines, inosines, xanthones, pteridines, 5-azacytidines (5-azacytidines), 5-azauracils (5-azauracils), triazolopyridines, imidazopyridines, pyrrolopyrimidines, pyrazolopyrimidines, adenine, N ⁶ Alkylpurine, N ⁶ Benzyl purine, N ⁶ Halogenated purines, N ⁶ Vinyl purine (vinylpurine), N ⁶ Acetyl purine, N ⁶ -acylpurines, N ⁶ Hydroxyalkyl purines, N ⁶ Thioalkylpurine, thymine, cytosine, 6-azapyrimidine, 2-mercaptopyrimidine, uracil, N ⁵ Alkylpyrimidines, N ⁵ -benzyl pyrimidine, N ⁵ -halogenated pyrimidines, N ⁵ Vinyl pyrimidine, N ⁵ Acetyl pyrimidine, N ⁵ -acyl pyrimidines, N ⁵ Hydroxyalkyl purines and N ⁶ -thioalkyl purines and isoxazolyl. The heteroaromatic group may be optionally substituted as described above for aryl. Heterocyclic or heteroaromatic groups may optionally be substitutedOne or more substituents selected from the group consisting of halogen, haloalkyl, alkyl, alkoxy, hydroxy, carboxy derivatives, amido, amino, alkylamino, and dialkylamino. The heteroaromatic compound may be partially or fully hydrogenated as desired. As a non-limiting example, dihydropyridines may be used in place of pyridines. Functional oxygen and nitrogen groups on the heterocycle or heteroaryl may be protected as needed or desired. Suitable protecting groups are well known to those skilled in the art and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl and t-butyldiphenylsilyl, trityl or substituted trityl, alkyl, acyl groups such as acetyl and propionyl, methanesulfonyl and p-toluenesulfonyl. The heterocyclic or heteroaromatic group may be substituted with any moiety that does not adversely affect the reaction, including but not limited to those described above for aryl groups.

The term "host" as used herein refers to a single-or multicellular organism to which the compound is administered, including but not limited to cell lines and animals, and preferably humans. The term host refers specifically to primates (including but not limited to chimpanzees) and humans. In most animal applications of the invention, the host is a human. However, in certain indications, the present invention clearly contemplates veterinary applications (e.g., for treating chimpanzees).

The term "peptide" refers to a natural or synthetic compound containing from 2 to 100 amino acids linked by the carboxyl group of one amino acid to the amino group of another amino acid.

The term "pharmaceutically acceptable salt or prodrug" is used throughout the specification to describe any pharmaceutically acceptable form (e.g., ester) of a compound that, upon administration to a patient, provides the compound. Pharmaceutically acceptable salts include salts derived from pharmaceutically acceptable inorganic bases and inorganic acids or organic bases and organic acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, and many other acids well known in the pharmaceutical arts.

Pharmaceutically acceptable prodrugs refer to compounds which are metabolized (e.g., hydrolyzed or oxidized) in the host to form the compounds of the invention. Typical examples of prodrugs include compounds having a biologically labile protecting group on the functional moiety of the active compound. Prodrugs include compounds that may be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrated, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. The prodrug forms of the compounds of the present invention may have antiviral activity, may be metabolized to form compounds that exhibit such activity, or both.

Non-limiting examples of phosphate/phosphonate prodrugs are described in the following references: ho, D.H.W. (1973) "Distribution of Kinase and deaminase of-beta-D-arabinofuranosylcytosine in tissues of man and muse." Cancer Res.33,2816-2820; holy, A. (1993) Isopolar phosphorous-modified nucleotide analogues, "In: de Clercq (Ed.), advances In Antiviral Drug Design, vol.I, JAIPress, pp.179-231; hong, c.i., nechaev, a. And West, c.r. (1979 a) "-Synthesis and antitumor activity of-beta-D-arabino-furanosylcytosine conjugates of cortisol and cobistone," bicohem.biophys.rs.commun.88,1223-1229; hong, c.i., nechaev, a., kirisits, A.J.Buchheit, D.J. and West, c.r. (1980) "Nucleoside conjugates as potential antitumor agents.3.Synthesis and antitumor activity of- (beta-D-arabinofuranosyl) cytosine conjugates of corticosteroids and selected lipophilic alcohos," j.med.chem.28,171-177; hosteller, K.Y., stuhmililer, L.M., lenting, H.B.M.van den Bosch, H.and Richman J.biol.chem.265,6112-6117; hosteller, K.Y., carson, D.A., and Richman, D.D. (1991); "Phosphatidylazidothidine: mechanism of antiretroviral action in CEM cells", "J.Biol chem.266,11714-11717; hosteller, K.Y., korba, B.Sridhar, C., gardner, M (1994 a) "Antiviral activity of phosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice." anti-viral Res.24,59-67; hosteller, K.Y., richman, D.D., sridhar.C.N.Felgner, P.L.Felgner, J., ricci, J., gardner, M.F.Selleseth, D.W. and Ellis, M.N. (1994 b) "Phosphatidylazidothymidine and phosphatidyl-ddC: assessment of uptake in mouse lymphoid tissues and antiviral activities in human immunodeficiency virus-infected cells and in rauscher leukemia virus-fed chemical," Antimicrobial Agents chemther.38, 2792-2797; hunston, r.n., jones, a.a. mcguilan, c., walker, r.t., balzarini, j. And DeClercq, e (1984) "Synthesis and biological properties of some cyclic phosphotriesters derived from' -deoxy-5-flouroudine," j.med.chem.27,440-444; ji, y.h., moog, c., schmitt, g., bisdoff, p., and Luu, b. (1990); monophosphoric acid esters of7- & gt beta-hydroxycholesterol and of pyrimidine nucleoside as potential antitumor agents: synthesis and preliminary evaluation of antitumor activity- & gt J.Med. Chem.332264-2270; jones, A.S., mcGuigan, C., walker, R.T., balzarini, J.and DeClercq, E. (1984) "Synthesis, properties, and biological activity of some nucleoside cyclic phosphamidate," J.chem.Soc.Perkin Trans.I., 1471-1474; juodka, B.A. and Smrt, J. (1974) "Synthesis of diribonucleoside phosph (P.fwdarw.N.) amino acid derivatives," Coll.Czech.chem.Comm.39,363-968; kataoka, s., imai, j., yamaji, n., kato, m., saito, m., kawada, t., and Imai, s. (1989) "Alkylated cAMP derivatives; selective synthesis and biological activites "-Nucleic Acids Res.Sym.Ser.21,1-2; kataoka, s., uchida, "(cAMP) benzyl and methyl triesters," Heterocycles 32,1351-1356; kinchington, D., harvey, J.J., O' Connor, T.J., jones, B.C.N.M., devine, K.G., taylor-Robinson D., jeffries, D.J., and McGuigan, C. (1992) "Comparison of Antiviral effects of zidovudine phosphoramidate an dphosphorodiamidate derivates against HIV and ULV in vitro," anti chem.chemther.3, 107-112; kodama, K., morozumi, M., saithoh, K.I., kuninaka, H., yosino, H., and Sanyoshi, M (1989) "Antitumor activity and pharmacology of-, -D-arabinofurosyl-5' -stearylphosphochate; an orally active derivative of1 beta-D-arabinofurosyl cytosine, "Jpn.J. cancer Res.80,679-685; korty, m. and Engels, j. (1979) "The effects of adenosine-and guanosine 3',5'phosphoric and acid benzyl esters on guinea-pic transverse myocard ium." Naunyn-schmiedberg ' sarch.pharmacol.310,103-111; kumar, a., goe, p.l., jones, A.S.Walker, R.T.Balzarini, j. And DeClercq, e (1990) "Synthesis and biological evaluation of some cyclic phosphoramidate nucleoside derivative," j.med.chem,33,2368-2375; leBec, c. and Huynh-Dinh, t. (1991) "Synthesis of lipophilic phosphate triester derivatives of 5-fluorouridine an arabinocytidine as anticancer pro-drugs" Tetrahedron lett.32,6553-6556; lichtenstein, j., barner, h.d., and Cohen, s.s. (1960) "The metabolism of exogenously supplied nucleotides by Escherichia coll.," j.biol.chem.235,457-465; luchy, j., von dantiken, a, friederich, j.manthey, b., zweifel, j., schlatter, c, and Benn, m.h. (1981) "Synthesis and toxicological properties of three naturally occurring cyanoepithioalkanes". Mitt.geg. lebensmittelunits.hyg. 72,131-133 (chem. Abstr.95, 127093); mcGigan, C.Tollerfield, S.M. and Riley, p.a. (1989) "Synthesis and biological evaluation of some phosphate triester derivatives of the antiviral drug ara." Nucleic Acids res.17,6065-6075; mcguilgan, c., devine, k.g., O ' Connor, t.j., galpin, s.a., jeffries, d.j., and Kinchington, d. (1990 a) "Synthesis and evaluation of some novel phosphoramidate derivatives of ' -azido-3' -deoxythymidine (AZT) as anti-HIV compositions," anti chem. Chemther.1-113; mcGuigan, C., O' Connor, T.J., nichols, S.R. Nickson, C.and Kinchington, D. (1990 b) "Synthesis and anti-HIV activity of some novel substituted dialkyl phosphate derivatives of AZT and ddCyd." Antiviral chem.chemther.1, 355-360; mcguilgan, c., nichols, s.r., O 'Connor, t.j. And Kinchington, d. (1990 c) "Synthesis of some novel dialkyl phosphate derivative of 3' -modified nucleosides as potential anti-AIDS drugs," anti-virus chem. Chemother.1,25-33; mcguilgan, c., devin, k.g., O ' Connor, t.j. And Kinchington, d. (1991) "Synthesis and anti-HIV activity of some haloalkyl phosphoramidate derivatives of 3' -azido-3' -deoxythylmidine (AZT); potent activity of the trichloroethyl methoxyalaninyl component "anti Res.15,255-263; mcguilgan, c., pathiran, r.n., balzarini, j. And DeClercq, e (1993 b) "Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of azt." j.med.chem.36,1048-1052.

Active compounds in one embodiment, the compounds have the following general formula:

or a pharmaceutically acceptable salt or prodrug thereof.

In this formula:

one of X and Z is selected from the group consisting of: -NH-, -N (NH) ₂ )-、-NH(OH)-、N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -, -O-, -CH ₂ -、-CH(C _1-10 Alkyl) -, C (C) _1-10 Alkyl group ₂ -、-CH(C _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ )-、-CH(OH)-、-CH(O-C _1-10 Alkyl) -, CH (NH) ₂ )-、-CH(NH-C _1-10 Alkyl) -and-CH (C (O) NH ₂ )-，

And the other of X and Z is selected from the group consisting of: -C (O) -SO ₂ -、-N(C(O)-、-CH ₂ -、-CH(C _1-10 Alkyl) -, C (C) _1-10 Alkyl group ₂ -、-CH(C _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ )-、-CH(NHC _1-10 Alkyl) -and-CH (C (O) NH ₂ )-，

Y is selected from the group consisting of: -NH, -N (NH) ₂ )-、-NH(OH)-、N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -, -O-, -CH ₂ -、-CH(C _1-10 Alkyl) -, CH (C) _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -C (C) _1-10 Alkyl group ₂ -、-CF ₂ -、-CCl ₂ -、-CH(CF ₃ )-、-CH(OH)-、-CH(O-C _1-10 Alkyl) -, -C (O) -, -SO ₂ -、-N(C(O)-C _1-10 Alkyl) -, -N (C (O) O-C _1-10 Alkyl) -, CH (NH) ₂ )-、-CH(NH-C _1-10 Alkyl) -and-CH (C (O) NH ₂ )-，

A and B are independently phenyl, a five membered heteroaromatic ring containing one, two or three nitrogen, oxygen or sulfur atoms, or a six membered heteroaromatic ring containing one, two or three nitrogen atoms;

u and v are independently 0, 1, 2, 3 or 4; provided that at least one of u and v is 1, 2, 3 or 4;

each R ¹ And R is ² Independently R is ³ 、OH、OR ³ 、SR ³ 、S(O)R ³ 、SO ₂ R ³ 、C(O)R ³ 、C(O)OR ³ 、OC(O)R ³ 、OC(O)OR ³ 、NH ₂ 、NHR ³ 、NHC(O)R ³ 、NR ³ C(O)R ³ 、NHS(O) ₂ R ³ 、NR ³ S(O) ₂ R ³ 、NHC(O)OR ³ 、NR ³ C(O)OR ³ 、NHC(O)NH ₂ 、NHC(O)NHR ³ 、NHC(O)N(R ³ ) ₂ 、NR ³ C(O)N(R ³ ) ₂ 、C(O)NH ₂ 、C(O)NHR ³ 、C(O)N(R ³ ) ₂ 、C(O)NHOH、C(O)NHOR ³ 、C(O)NHSO ₂ R ³ 、C(O)NR ³ SO ₂ R ³ 、SO ₂ NH ₂ 、SO ₂ NHR ³ 、SO ₂ N(R ³ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ³ 、C(N)N(R ³ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), -CH ₂ Phosphonates, -CH ₂ O-phosphate, CH ₂ P(O)(OH) ₂ 、CH ₂ P(O)(OR ³ ) ₂ 、CH ₂ P(O)(OR ³ )(NR ³ )、CH ₂ P(O)(NR ³ ) ₂ 、CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) or CH ₂ A cyclopSal monophosphate prodrug,

wherein the term phosphate includes mono-, di-, tri-and stabilized phosphate prodrugs, the term phosphonate includes the same prodrugs present in phosphate prodrugs,

and when R is ¹ And R is ² On adjacent carbons, they may together form a saturated or unsaturated alkyl, aromatic or heteroaromatic ring;

each R ³ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁴ 、OH、OR ⁴ 、SR ⁴ 、S(O)R ⁴ 、SO ₂ R ⁴ 、C(O)R ⁴ 、C(O)OR ⁴ 、OC(O)R ⁴ 、OC(O)OR ⁴ 、NH ₂ 、NHR ⁴ 、NHC(O)R ⁴ 、NR ⁴ C(O)R ⁴ 、NHS(O) ₂ R ⁴ 、NR ⁴ S(O) ₂ R ⁴ 、NHC(O)OR ⁴ 、NR ⁴ C(O)OR ⁴ 、NHC(O)NH ₂ 、NHC(O)NHR ⁴ 、NHC(O)N(R ⁴ ) ₂ 、NR ⁴ C(O)N(R ⁴ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁴ 、C(O)N(R ⁴ ) ₂ 、C(O)NHOH、C(O)NHOR ⁴ 、C(O)NHSO ₂ R ⁴ 、C(O)NR ⁴ SO ₂ R ⁴ 、SO ₂ NH ₂ 、SO ₂ NHR ⁴ 、SO ₂ N(R ⁴ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁴ 、C(N)N(R ⁴ ) ₂ 、C(N)OH、C(N)OCH ⁴ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O)OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl), cyclophosphate prodrugs, CH ₂ P(O)(OH) ₂ 、CH ₂ P(O)(OR ⁴ ) ₂ 、CH ₂ P(O)(OR ⁴ )(NR ⁴ )、CH ₂ P(O)(NR ⁴ ) ₂ 、CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and CH ₂ A cyclopSal monophosphate prodrug,

each R ⁴ Independently selected from aryl, heteroaryl, arylalkyl, alkylaryl, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl and C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁵ 、OH、OR ⁵ 、SR ⁵ 、S(O)R ⁵ 、SO ₂ R ⁵ 、C(O)R ⁵ 、C(O)OR ⁵ 、OC(O)R ⁵ 、OC(O)OR ⁵ 、NH ₂ 、NHR ⁵ 、NHC(O)R ⁵ 、NR ⁵ C(O)R ⁵ 、NHS(O) ₂ R ⁵ 、NR ⁵ S(O) ₂ R ⁵ 、NHC(O)OR ⁵ 、NR ⁵ C(O)OR ⁵ 、NHC(O)NH ₂ 、NHC(O)NHR ⁵ 、NHC(O)N(R ⁵ ) ₂ 、NR ⁵ C(O)N(R ⁵ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁵ 、C(O)N(R ⁵ ) ₂ 、C(O)NHOH、C(O)NHOR ⁵ 、C(O)NHSO ₂ R ⁵ 、C(O)NR ⁵ SO ₂ R ⁵ 、SO ₂ NH ₂ 、SO ₂ NHR ⁵ 、SO ₂ N(R ⁵ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁵ 、C(N)N(R ⁵ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br orI)、P(O)(OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

each R ⁵ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁶ 、OH、OR ⁶ 、SR ⁶ 、S(O)R ⁶ 、SO ₂ R ⁶ 、C(O)R ⁶ 、C(O)OR ⁶ 、OC(O)R ⁶ 、OC(O)OR ⁶ 、NH ₂ 、NHR ⁶ 、NHC(O)R ⁶ 、NR ⁶ C(O)R ⁶ 、NHS(O) ₂ R ⁶ 、NR ⁶ S(O) ₂ R ⁶ 、NHC(O)OR ⁶ 、NR ⁶ C(O)OR ⁶ 、NHC(O)NH ₂ 、NHC(O)NHR ⁶ 、NHC(O)N(R ⁶ ) ₂ 、NR ⁶ C(O)N(R ⁶ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁶ 、C(O)N(R ⁶ ) ₂ 、C(O)NHOH、C(O)NHOR ⁶ 、C(O)NHSO ₂ R ⁶ 、C(O)NR ⁶ SO ₂ R ⁶ 、SO ₂ NH ₂ 、SO ₂ NHR ⁶ 、SO ₂ N(R ⁶ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁶ 、C(N)N(R ⁶ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ 、F、Cl、Br、I、P(O)(OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

each R ⁶ Independently an aryl group, a heteroaryl group, C _3-10 NaphtheneRadical, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: c (C) _1-10 Alkyl, C _2-10 Alkenyl, C _2-10 Alkynyl, OH, NH ₂ 、C(O)NH ₂ 、C(O)NHOH、SO ₂ NH _2、 COOH、C(O)H、C(N)NH ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug.

Pharmaceutically acceptable salts and prodrugs of these compounds are also intended to be within the scope of the present invention.

Representative R ² The structural part is as follows:

representative R ³ The structural part is as follows:

in one embodiment, one of X and Z is-C (O) -, -SO ₂ -or-NC (O) -, and the other is-NH-, -N (NH) ₂ )-、-NH(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -or-O-.

In another embodiment, one of X and Z is-C (O) -, -SO ₂ -or-N (C (O) -, and the other is-CH ₂ -、-CH(C _1-6 Alkyl) -, C (alkyl) ₂ -、-CH(C _3-8 Cycloalkyl) -, CH (C) _2-6 Alkenyl, -CH (C) _2-6 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ ) -, -CH (NH alkyl) -or-CH (C (O) NH) ₂ )-。

In another embodiment, one of X and Z is-NH-, -N (NH) ₂ ) -, -NH (OH) -, -N (alkyl) -or-O-, and the other is-CH ₂ -、-CH(C _1-6 Alkyl) -, C (alkyl) ₂ -、-CH(C _3-8 Cycloalkyl) -, CH (C) _2-6 Alkenyl, -CH (C) _2-6 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ ) -, -CH (NH alkyl) -or-CH (C (O) NH) ₂ )-。

In a third embodiment, one of X and Z is-NH-, -N (NH) ₂ )-、-NH(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -, and the other is-C (O) -or-SO ₂ -。

In a fourth embodiment, Y is-NH, -N (NH) ₂ )-、-NH(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -or-O-.

In a fifth embodiment, Y is-NH, -N (NH) ₂ )-、-NH(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -,

in a sixth embodiment, R ¹ And R is ² One is H, -CH ₂ Phosphonates, -CH ₂ O-phosphates, wherein the term phosphate includes mono-, di-, tri-and stabilized phosphate prodrugs, and the term phosphonate includes the same prodrugs present in phosphate prodrugs.

In a seventh embodiment, R ¹ And R is ² One of them is H, -CH ₂ P(O)(OH) ₂ 、-CH ₂ P(O)(OH)(OR ⁶ )、-CH ₂ P(O)(OR ⁶ ) ₂ 、-CH ₂ P(O)(OR ⁶ )(NR ⁶ )、-CH ₂ P(O)(NR ⁶ ) ₂ 、-CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) or-CH ₂ -a cyclopsala monophosphate prodrug.

In one aspect of this embodiment, R ¹ And R is ² One of them is a phosphonate, phosphoramidate, cyclopsala monophosphate prodrug, or a compound having the general formula-CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl).

In a preferred embodiment, R ¹ And R is ² One of them is-C (O) NHR ⁴ 、-C(O)N(R ⁴ ) ₂ ，

Wherein R is ⁴ Is C _1-10 Alkyl, C _3-10 Cycloalkyl, C _2-10 Alkenyl, C _2-10 Alkynyl, arylalkyl, alkylaryl, C _1-10 Haloalkyl, C _1-10 Alkyl-aryl or C _1-10 Haloalkyl-aryl, m is 0, 1 or 2. In particular embodiments, R ⁴ Is C _1-10 Alkyl-aryl, and benzyl is particularly preferred R ⁴ A substituent.

In another embodiment, R ¹ And R is ² One of them is-C (O) -C _1-10 Alkyl, -C (O) -alkylaryl, -C (O) -heterocyclyl-CH ₂ -aryl, -C (O) -heterocyclyl-CF ₂ -aryl, -C (O) -cycloalkyl-alkylaryl, -C (O) NHC _1-10 Alkyl, -C (O) NH-alkylaryl, -C (O) NH-heterocyclyl-CF ₂ -aryl, -C (O) NH-cycloalkyl-alkylaryl, -SO ₂ -C _1-10 Alkyl, -SO ₂ -alkylaryl, -SO ₂ -heterocyclyl-alkylaryl, -SO ₂ -heterocyclyl-CF ₂ -aryl or-SO _2- Cycloalkyl-alkylaryl.

The specific variables shown above may also be used in connection with any of formulas B through H, as will be discussed in detail below.

Representative compounds include the following:

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or a pharmaceutically acceptable salt or prodrug thereof. Particularly preferred compounds have the general formula:

or a pharmaceutically acceptable salt or prodrug thereof. />

In other embodiments, the compound has one of the following formulas:

and pharmaceutically acceptable salts and prodrugs thereof, wherein R ² And u is as defined above for formula A, except that u may be 0.

One representative compound has the general formula:

and pharmaceutically acceptable salts and prodrugs thereof, wherein R ² And u is as defined above for formula A, except that u may be 0.

In another embodiment, the compound has one of the following formulas:

/>

/>

and pharmaceutically acceptable salts or prodrugs thereof, wherein R ² And u is as defined above for formula A, except that u may be 0 and n is 0, 1 or 2.

Stereoisomerism and polymorphism

The compounds described herein may have asymmetric centers and may exist as racemates, racemic mixtures, individual diastereomers or enantiomers, all isomeric forms being included in the present invention. The compounds of the invention having chiral centers may exist and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. The present invention includes racemic, optically-active, polymorphic, or stereoisomeric forms, or mixtures thereof, of the compounds of the present invention having the useful properties described herein. The optically active form can be prepared, for example, by: resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis or by chromatographic separation using a chiral stationary phase, or by enzymatic resolution. The corresponding compound may be purified and then the compound is derivatized to form the compounds described herein, or the compound itself may be purified.

The optically active form of the compound may be prepared using any method known in the art, including, but not limited to, resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.

Examples of the method of obtaining an optically active material include at least the following methods:

i)physical separation of crystals:techniques for manual separation of macroscopic crystals of individual enantiomers. This technique can be employed if crystals of individual enantiomers are present, i.e. the starting material is an aggregate (agglomerate) and the crystals are visually distinguishable;

ii)simultaneous crystallization:techniques for crystallizing individual enantiomers separately from solutions of racemates may be feasible only when the racemates are solid aggregates;

iii)enzymatic resolution method:techniques for partially or completely separating racemates by means of different rates of enantiomer reaction with enzymes;

iv)enzymatic asymmetric synthesis:at least one synthesis step employs a synthesis technique employing an enzymatic reaction to obtain enantiomerically pure or enriched synthesis precursors of the desired enantiomer;

v)chemical asymmetric synthesis method: Synthesis techniques for synthesizing the desired enantiomer from an achiral precursor under conditions that produce asymmetry (i.e., chirality) in the product, which can be accomplished using chiral catalysts or chiral auxiliary;

vi)diastereoisomeric separation process:techniques for converting individual enantiomers to diastereomers by reacting a racemic compound with an enantiomerically pure reagent (chiral auxiliary). The diastereomers thus obtained are then separated by chromatography or crystallization, with the now more pronounced structural differences, followed by removal of the chiral auxiliary to obtain the desired enantiomer;

vii)primary and secondary asymmetric conversion process:this technique breaks this equilibrium by balancing the diastereomers from the racemates so that they are dominant in the solution of the diastereomers from the desired enantiomer, or preferential crystallization of the diastereomers from the desired enantiomer, such that all material is ultimately converted almost to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereoisomer;

viii)kinetic resolution method:the technology refers to the realization of partial or complete resolution of racemates (or further resolution of partially resolved compounds) by means of different reaction rates of enantiomers with chiral, non-racemic reagents or catalysts under kinetic conditions;

ix)Enantiomer specific synthesis from non-racemic precursors:synthetic techniques that obtain the desired enantiomer from a non-chiral starting material and that do not, or only minimally, disrupt the stereochemical integrity during synthesis;

x)chiral liquid chromatography:techniques for separating enantiomers of racemates in liquid mobile phases by virtue of their different interactions with stationary phases, including but not limited to by chiral HPLC. The stationary phase may be made of chiral material or the mobile phase may contain additional chiral material that causes different interactions;

xi)chiral gas chromatography:techniques for separating enantiomers by volatilizing the racemates and by means of different interactions of the enantiomers in a gaseous mobile phase with a column containing a fixed non-racemic chiral adsorption phase;

xii)extraction with chiral solvent:a technique for separating enantiomers by preferential dissolution of one enantiomer in a specific chiral solvent;

xiii)trans-chiral membrane transport method:techniques for contacting racemates with thin film barriers. The barrier typically separates two miscible liquids, one of which contains racemates, with a driving force such as a concentration differential or pressure differential resulting in preferential transport across the membrane barrier. The non-racemic chiral nature of the membrane allows only one enantiomer of the racemate to pass through, thereby effecting separation.

Chiral chromatography is used in one embodiment, including but not limited to simulated moving bed chromatography. Various chiral stationary phases are commercially available.

IV salt or prodrug formulations

Where the compound is sufficiently basic or acidic to form a stable non-toxic acid or base salt, it may be appropriate to administer the compound as a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form physiologically acceptable anions such as tosylate, mesylate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, alpha-ketoglutarate and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including, but not limited to, sulfate, nitrate, bicarbonate, and carbonate. For certain transdermal applications, it may be preferable to use fatty acid salts of the compounds described herein. Fatty acid salts can help penetrate the stratum corneum. Examples of suitable salts include salts of the compounds with stearic acid, oleic acid, linoleic acid, palmitic acid, caprylic acid and capric acid.

Pharmaceutically acceptable salts can be obtained using standard methods well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid (providing a physiologically acceptable anion). In those cases where the compound includes a plurality of amine groups, salts may be formed with any number of amine groups. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids may also be prepared.

A prodrug is a pharmacological substance that is administered in an inactive (or significantly less active) form and subsequently metabolized in vivo to an active metabolite. Achieving more drug to the desired target at lower doses is often the rationale behind the use of prodrugs and this is often due to better absorption, distribution, metabolism and/or excretion (ADME) properties. Prodrugs are often designed to improve oral bioavailability because malabsorption in the gastrointestinal tract is often the limiting factor. In addition, the use of prodrug strategies can increase the selectivity of a drug for its intended target, thereby reducing the likelihood of off-target effects.

V. therapeutic methods

The host may be treated by administering to the patient an effective amount of the active compound or a pharmaceutically acceptable prodrug or salt thereof, optionally in the presence of a pharmaceutically acceptable carrier or diluent. The active substance may be administered by any suitable route, for example orally, parenterally, intravenously, intradermally, transdermally, subcutaneously or topically in liquid or solid form. Details of administration are provided in the pharmaceutical compositions.

The compounds are useful for treating, preventing, reducing the susceptibility to, reducing the severity of, or delaying the progression of pancreatitis, sarcopenia, stroke, and traumatic brain injury associated with FGF21, as well as glioblastoma and other disorders associated with miR-122. In some embodiments, the compounds are administered with other agents for treating these disorders.

Treatment of glioblastoma

Glioblastoma (GBM) is an almost fatal brain tumor that requires large amounts of free Fatty Acids (FAs) to promote cell growth. Although high levels of FAs often lead to lipotoxicity, studies have shown that GBM avoids lipotoxicity by up-regulating diacylglycerol-acyltransferase 1 (DGAT 1) to store excess FAs in the form of triglycerides and lipid droplets (Cheng et al, 2020,Cell Metabolism 32,229-242 (8, 4, 2020)).

Cheng regards inhibition of DGAT1 as a way to disrupt the balance within liposomes and indicates that inhibition of DGAT1 results in excess FAs entering mitochondria for oxidation. This results in the production of high levels of Reactive Oxygen Species (ROS), mitochondrial damage, cytochrome c release, and tumor cell apoptosis. In animal models of GBM, cheng indicated that targeting DGAT1 blocked lipid droplet formation, induced tumor cell apoptosis, and significantly inhibited GBM growth, and indicated that targeting DGAT1 could be a promising approach to GBM treatment. However, DGAT1 inhibitors are associated with adverse gastrointestinal events such as nausea, diarrhea and vomiting (DeVita and Pinto, "Current status of the research and development of diacylglycerol O-Acyltransferase 1 (DGAT 1) inhibitors," J Med chem.56 (24): 9820-5 (2013), and DGAT1 inhibitors evaluated by Cheng (DGAT 1 inhibitor A-922500) are unable to cross the blood brain barrier and therefore would not be able to treat GBM.

There is a correlation between miR-122 and lipid droplet formation (Wu et al, "MicroRNA-122Inhibits Lipid Droplet Formation and Hepatic Triglyceride Accumulation via Yin Yang 1," Cell Physiol biochem.,44 (4): 1651-1664 (2017)). Wu discloses that intracellular lipid droplet formation and increased liver Triglyceride (TG) content can lead to nonalcoholic fatty liver disease, miR-122 down-regulation in Free Fatty Acid (FFA) induced steatosis hepatocytes, and streptozotocin and high fat diet (STZ-HFD) induced non-alcoholic steatohepatitis (NASH) in mice. Transfection of hepatocytes with miR-122 mimics prior to FFA induction inhibited lipid droplet formation and TG accumulation in vitro.

The ROR-alpha agonist compounds described herein increase circulating miR-122 levels. Since miR-122 can cross the blood-brain barrier, administration of these compounds increases miR-122 levels in the brain. Thus, the compounds can block lipid droplet formation, induce apoptosis in tumor cells, and inhibit GBM growth.

VI combination or alternation therapy

In one embodiment, the compound of formula (a) or a pharmaceutically acceptable derivative thereof may be used alone, in combination with one or more compounds of formula (a) or a pharmaceutically acceptable derivative thereof, or in combination with at least one other agent for treating conditions associated with ROR.

In certain embodiments, compounds of formula (a) for use in treating pancreatitis are combined with agents such as, but not limited to, analgesics (e.g., acetaminophen ibuprofen, hydrocodone, tramadol, or naproxen) enzyme pellets that aid digestion, vitamins (e.g., vitamins A, B, D, E, and/or K if the patient suffers from malabsorption)) and/or STAT3 (signal transducer and transcriptional activator) inhibitors (e.g., niclosamide, WP1066 (WPD pharmaceutical), OPB-51602 (medko Biosciences)) and inhibitors of other members of the STAT protein family (including STAT1, STAT2, STAT4, STAT5A and STAT 5B), and STAT 6).

The course of treatment may be tracked, for example, blood tests to find elevated pancreatin levels, stool tests in chronic pancreatitis to measure fat levels that may suggest that the patient's digestive system is not sufficiently absorbing nutrition, computed Tomography (CT) scans to find gall stones and evaluate the extent of pancreatic inflammation, abdominal ultrasound to find gall stones and pancreatic inflammation, endoscopic ultrasound to find inflammation and blockage in pancreatic ducts or bile ducts, and/or Magnetic Resonance Imaging (MRI) to find abnormalities in the gall bladder, pancreas, and ducts.

When used to treat sarcopenia, the compounds may be co-administered with urocortin II, hormones (such as testosterone or growth hormone), STAT3 inhibitors (such as niclosamide, WP1066 (WPD pharmaceutical), OPB-51602 (medko Biosciences) and S3I-201 (Santa Cruz Biotechnology)), and inhibitors of other members of the STAT protein family (including STAT1, STAT2, STAT4, STAT5 (STAT 5A and STAT 5B) and STAT 6), and drugs for the treatment of metabolic syndrome (including insulin resistance, obesity and hypertension) (metformin and other AMPK agonists).

When used to treat glioblastoma, the compounds may be administered with other treatments for glioblastoma. One or more of the other active agents described below, in any combination, may be administered to positively treat GBM.

Treatment of glioblastomas has been very difficult in the past due to several complex factors. Tumor cells are very resistant to traditional therapies, the brain is vulnerable to traditional therapies, the ability to self-repair is very limited, and many drugs cannot act on tumors across the blood brain barrier.

Temozolomide (TMZ) is one example of a drug that may be used to treat glioblastoma multiforme. It can be administered orally or intravenously.

Cannabinoids, whether in the form of Tetrahydrocannabinol (THC), synthetic analogues of nabilone (nabilone), CBD, CBG or other cannabinoids, may be co-administered. These compounds are useful against chemotherapy-induced nausea and vomiting, stimulating appetite, alleviating pain and pain, and inhibiting the growth and angiogenesis of malignant gliomas. Cannabinoids can attack the tumor stem cells of glioblastomas, inducing them to differentiate into more mature (and therefore more "treatable") cells.

Berberine, an isoquinoline alkaloid, is an example of a compound that may be co-administered. The anti-tumor effect of berberine on glioblastoma cells is thought to involve induction of cellular senescence, inhibition of the RAF-MEK-ERK signaling pathway, and/or down-regulation of EGFR.

Direct nasal-brain drug delivery can be used to achieve higher and desirably more effective drug concentrations of the compounds described herein, as well as other active agents described herein, in the brain. The natural compound perillyl alcohol may be administered intranasally, for example as an aerosol.

GBM tumors contain areas of tissue that exhibit hypoxia, which is highly resistant to radiation therapy. The radiosensitizer may be co-administered with radiation therapy. Oxygen diffusion enhancing compounds such as sodium trans crocetin are examples of radiosensitizers. Boron neutron capture therapy has been tested as an alternative therapy to glioblastoma.

Anticonvulsants such as sodium phenytoin may be co-administered, particularly after seizures. Corticosteroids (usually dexamethasone) can reduce peri-cancerous oedema (rearrangement through the blood brain barrier), reduce mass effects, lower intracranial pressure, reduce headache or sleepiness.

The compounds described herein may also be combined with Chimeric Antigen Receptor (CAR) T cell therapies. CAR T cells using CLTX as the targeting domain (CLTX-CAR T cells) mediate potent anti-GBM activity and effectively target tumors lacking expression of other GBM-related antigens (Wang et al, "Chlorotoxin-directed CAR T cells for specific and effective targeting of glioblastoma," Science Translational Medicine, vol.12, issue 533, eaaw2672 (3 months 2020)). CAR T cell therapies using IL13 ra 2, her2/CMV, EGFRvIII, CSPG4, NKG2DL, CD19 and CD133 as targeting domains can also be used. Representative therapies include kymeriah and yescanta of Novartis and Gilead Sciences.

MP-Pt (IV) is a MAOB sensitive mitochondrial-specific prodrug for the treatment of glioblastoma, which may also be combined with the compounds described herein.

RIPGBM (N- [1, 4-dihydro-1, 4-dioxo-3- [ (phenylmethyl) amino ] -2-naphthyl ] -N- [ (4-fluorophenyl) methyl ] acetamide) is a RIPK2 modulator and is a prodrug of crpgm. The compounds selectively induce apoptosis in glioblastoma multiforme cancer stem cell lines and are orally bioavailable and brain penetrating. The structure is as follows:

inhibitors of cyclin D1/CDK4 and CDK6 are another anti-cancer agent that may be combined with the compounds described herein.

In view of the invasive nature of GBM, it is contemplated that one or more of these active agents may be combined with the compounds described herein to attack GBM through a variety of biological pathways.

When used to treat traumatic brain injury, the compounds may be co-administered with tranexamic acid (administered shortly after injury), sedatives, analgesics, and paralyzing agents, while controlling intracranial pressure (ICP), antiepileptics, such as phenytoin and levetiracetam (levitetam), and norepinephrine or similar drugs that help maintain cerebral perfusion, intranasal insulin (as described in us patent No. 10,314,911), and VLA-1 (very late activating antigen-I) antagonists.

When used in the treatment of stroke, the compounds may be co-administered with a compound that inhibits clot formation (e.g., a blood diluent) or a compound that breaks down an existing clot (e.g., tissue Plasminogen Activator (TPA), eptifibatide, acipimab (ReoPro), or tirofiban (Aggrastat)).

The blood thinner prevents blood clot formation and prevents existing blood clots from becoming larger. There are two main blood diluents. Anticoagulants such as heparin or warfarin (also known as coumarin) slow down the biological processes that produce blood clots, and antiplatelet agents such as brivia, aspirin prevent blood cells called platelets from aggregating together to form blood clots.

For example, the processing steps may be performed,generally byA dose of 180mcg/kg was administered as a bolus intravenous injection as soon as possible after diagnosis, with a continuous infusion (after initial bolus) of 2mcg/kg/min for up to 96 hours of treatment.

Representative platelet aggregation inhibitors include glycoprotein IIB/IIIA inhibitors, phosphodiesterase inhibitors, adenosine reuptake inhibitors, and Adenosine Diphosphate (ADP) receptor inhibitors. These may optionally be administered in combination with an anticoagulant.

Representative anticoagulants include coumarins (vitamin K antagonists), heparin and its derivatives (including normal heparin (UFH), low Molecular Weight Heparin (LMWH) and Ultra Low Molecular Weight Heparin (ULMWH)), synthetic pentosan inhibitors of factor Xa (including Fondaparinux, idaparin (Idraparinux) and idrabioaparinux), direct acting oral anticoagulants (DAOCs) (e.g., dabigatran, rivaroxaban, apixaban, edoxaban and betrofiban), and antithrombin protein therapeutics/thrombin inhibitors (e.g., the bivalent drugs hirudin, recombinant hirudin and bivalirudin and monovalent argatroban).

Representative platelet aggregation inhibitors include pravastatin, plamix (clopidogrel bisulfate), platal (cilostazol), effect (prasugrel), aggrenox (aspirin and dipyridamole), briinta (ticagrelor), carboxizumab (cappucizumab), kentreal (cangrel), dipyridamole, ticlopidine (ticlopidine), yopprala (aspirin and omeprazole).

The compounds may also be combined with neuroprotective agents (e.g., thrombolytics), erythropoiesis stimulating agents (e.g., erythropoietin, dapoxetine and erythropoietin alpha), ET _B Receptor agonists (e.g., IRL-1620), ET _A Receptor agonists (e.g., sulfonylisoxazole, cladribine, atrasentan, tizosentan, bosentan, sitaxsentan, enradsentan, BMS207940, BMS193884, BMS182874, J104132, VML 588/Ro 61 1790, T-0115, TAK 044, BQ 788, TBC2576, TBC3214, PD180988, ABT 546, SB247083, RPR118031A and BQ 123), and Argatroban, alfimeprase, tenecteplase, ancrod, sildenafil, insulin growth factor, magnesium sulfate, human serum albuminThe co-administration of caffeine alcohol, microphospholipidase, statin, eptifibatide, etanercept, citicoline, edaravone, cilostazol, and mixtures thereof.

Other drugs used in combination for ROR-related conditions include, but are not limited to, the following: cholesterol biosynthesis inhibitors (HMG CoA reductase inhibitors such as lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, nilvastatin and rivastatin); squalene epoxidase inhibitors (e.g., terbinafine); plasma HDL-raising agents (e.g., CETP inhibitors such as ansetronpi, R1658); human peroxisome proliferator-activated receptor (PPAR) gamma agonists (e.g., thiazolidinediones such as rosiglitazone, troglitazone and pioglitazone); pparα agonists (e.g., clofibrate, fenofibrate, and Ji Feibei ni); PPAR bi-alpha/gamma agonists (e.g., mogliclazide (muraglitazar), alglitazide (aleglitazar), peglitazide (peliglitazazar), elafilbar); farnesoid X Receptor (FXR) modulators (e.g., obeticholic acid, LMB763, LJN45, etc.); bile acid sequestrants (e.g., anion exchange resins or quaternary amines (e.g., cholestyramine or colestipol); bile Acid Transport Inhibitors (BATi); nicotinic acid, nicotinamide; cholesterol absorption inhibitors (e.g., ezetimibe); acyl-coa cholesterol O-acyl transferase (ACAT) inhibitors (e.g. avastin); selective estrogen receptor modulators (e.g., raloxifene or tamoxifen); lxrα or β agonists, antagonists or partial agonists (e.g., 22 (R) -hydroxycholesterol, 24 (S) -hydroxycholesterol, T0901317 or GW 3965); microsomal triglyceride transfer protein (MTP) inhibitors, antidiabetic agents such as insulin and insulin analogues (e.g., lysPro insulin, inhalant formulations containing insulin; sulfonylureas and analogues (e.g., tosylamide, chlorpropionyl urea, glipizide, glimepiride, glibenclamide, acetosulfourea, glipizide), biguanides (e.g., metformin or metformin hydrochloride, phenformin, buformin) alpha 2 antagonists and imidazolines (e.g., miglitazone), idazol (isaglidole), deglidole (deglidole), imidazoloxan (idazoxan), efaciens (efaroxan), flucloxacin (fluxaroxan)), thiazolidinediones (e.g., pioglitazone hydrochloride, rosiglitazone maleate, cilaglitazone, troglitazone or baglione), alpha-glucosidase inhibitors (e.g., miglitol, acarbose, eparata or voglibose), meglitinides (e.g., repaglinide or nateglinide), DPP-4 inhibitors (e.g., sitagliptin phosphate, saxagliptin, vildagliptin, alogliptin or deglutamine), incretin (e.g., glucagon-like peptide-1 (GLP-1) receptor agonists (e.g., exenatide) (bytta) ^TM ) NN2211 (liraglutide), GLP-1 (7-36) amide and analogues thereof, GLP-1 (7-37) and analogues thereof, AVE-0010 (ZP-10), R1583 (tasmaglutide), GSK-716155 (Abirutate, GSK/human genome science), BRX-0585 (pyro/Biorexis) and CJC-1134-PC (Exendin-4: PC-DAC) ^TM And glucose-dependent insulinotropic peptide (GIP)); islet amyloid polypeptide agonists (e.g., pramlintide, AC-137); insulin secretagogues (e.g., lignogliride, nateglinide, repaglinide, mitiglinide calcium hydrate (mitiglinide calcium hydrate) or meglitinide); SGLT-2 inhibitors (e.g., dapagliflozin (BMS), sertacolin (Kissei), AVE 2268 (Sanofi-Aventis)), glucokinase activators (e.g., compounds disclosed in WO 00/58293A 1), anti-obesity agents such as nerve growth factor agonists (e.g., axokine), growth hormone agonists (e.g., AOD-9604), adrenergic uptake inhibitors (e.g., GW-320659), 5-HT (serotonin) reuptake/transport inhibitors (e.g., fluoxetine (Prozac)), 5-HT/NA (serotonin/norepinephrine) reuptake inhibitors (e.g., sibutramine), DA (dopamine reuptake inhibitors (e.g., bupropion), 5-HT, NA and DA reuptake blockers, steroidal plant extracts (e.g., P57), NPY1 or 5 (neuropeptide Y Y or Y5) antagonists, NPY2 (neuropeptide Y Y) agonists, MC4 (melanocortin 4) agonists, CCK-A (serotonin) and MCH-SR 1 receptor antagonists (e.g., MCH1R 41 receptor antagonists), and anti-melanin receptor (e.g., melanin receptor (GHR 1) antagonists, 41 receptor (e.g., anti-melanin receptor 41) antagonists) R) agonists/antagonists, H3 (histamine receptor 3) inverse agonists or antagonists, H1 (histamine 1 receptor) agonists, FAS (fatty acid synthase) inhibitors, ACC-1 (acetyl CoA carboxylase-1) inhibitors, beta 3 (beta adrenergic receptor 3) agonists, DGAT-2 (diacylglycerol acyltransferase 2) inhibitors, DGAT-1 (diglycerol acyltransferase 1) inhibitors, CRF (corticotropin releasing factor) agonists, galanin antagonists, UCP-1 (uncoupling protein-1), 2 or 3 activators, leptin or leptin derivatives, opioid antagonists, orexin antagonists, BRS3 agonists, GLP-1 (glucagon-like peptide 1) agonists, IL-6 agonists, alpha-MSH agonists, agRP antagonists, BRS3 (bombesin receptor subtype 3) agonists, 5-HT1B agonists, POMC antagonists, CNTF (ciliated neurotrophins or CNTF derivatives), NN2211, topiramate, glucocorticoid antagonists, exenatide-4 agonists, 5-HT2C (serotonin receptor 2C) agonists (e.g. Lorcaserin), PDE (phosphodiesterase) inhibitors, fatty acid transporter inhibitors, dicarboxylic acid ester transporter inhibitors, glucose transporter inhibitors, CB-1 (cannabinoid 1 receptor) inverse agonists or antagonists (e.g. SR 141716), lipase inhibitors (e.g., orlistat); cyclooxygenase-2 (COX-2) inhibitors (e.g., rofecoxib and celecoxib); thrombin inhibitors (e.g., heparin, argatroban, melagatran, dabigatran); platelet aggregation inhibitors (e.g., glycoprotein IIb/IIIa fibrinogen receptor antagonists or aspirin); vitamin B6 and pharmaceutically acceptable salts thereof; vitamin B12; vitamin E; folic acid or a pharmaceutically acceptable salt or ester thereof; antioxidant vitamins, such as C, E and beta-carotene; beta blockers (e.g., angiotensin II receptor antagonists such as losartan, irbesartan or valsartan; angiotensin converting enzyme inhibitors such as enalapril and captopril; calcium channel blockers such as nifedipine and diltiazem; endothelial antagonists; aspirin; fatty acid/bile acid complexes (Aramichol); caspase inhibitors (emricasan); immunomodulators (Cenicriviroc et Al); thyroid hormone receptor modulators (MB 07811, MGL-3196, etc.); ATP-binding cassette transporter-Al gene expression enhanced other than LXR ligands) A medicament; and bisphosphonate compounds (e.g., alendronate sodium).

In certain embodiments, the compound of formula (a) is combined with at least one other agent that modifies host metabolism, such as, but not limited to, clarithromycin, cobicistat, indinavir, itraconazole, ketoconazole, nefazodone, ritonavir, saquinavir, shu Beisheng (suloxone), telithromycin, aprepitant, erythromycin, fluconazole, verapamil, diltiazem, cimetidine, amiodarone, boceprevir (boceprevir), chloramphenicol, ciprofloxacin, delavirdine, diethyl-dithiocarbamate, fluvoxamine, gestodene, imatinib, mi Beide l, mifepristone, norfloxacin, norfluoxetine, telaprevir, and voriconazole.

VII pharmaceutical composition

The host may be treated by administering to the patient an effective amount of the active compound or a pharmaceutically acceptable prodrug or salt thereof in the presence of a pharmaceutically acceptable carrier or diluent, including but not limited to humans suffering from pancreatitis, stroke, brain trauma, or sarcopenia. The active substance may be administered by any suitable route, for example orally, parenterally, intravenously, intradermally, subcutaneously or topically in liquid or solid form.

Preferred dosages of the compounds range from about 0.01 to about 10mg/kg, more typically from about 0.1 to 5mg/kg, and preferably from about 0.5 to about 2mg/kg of the recipient's body weight per day. The effective dosage range of the pharmaceutically acceptable salts and prodrugs can be calculated based on the weight of the parent compound to be delivered. If the salt or prodrug itself exhibits activity, the weight of the salt or prodrug can be used to estimate the effective dose as above, or by other methods known to those skilled in the art.

The compounds may conveniently be administered in any suitable dosage unit, including but not limited to dosage forms containing from 7 to 600mg, preferably from 70 to 600mg, of active ingredient per unit dosage form. An oral dosage of 5-400mg is generally convenient.

The concentration of the active compound in the pharmaceutical composition will depend on the absorption, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It should be noted that the dosage value will also vary with the severity of the condition to be alleviated. It will also be appreciated that the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, for any particular subject, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. The active ingredient may be administered at one time or may be divided into a number of smaller doses and administered at different time intervals.

The preferred mode of administration of the active compounds is oral. Oral compositions typically include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For therapeutic oral administration purposes, the active compounds may be combined with excipients and used in the form of tablets, troches or capsules. Pharmaceutically compatible binders and/or adjuvant materials may be included as part of the composition.

Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, primogel or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the type described above, a liquid carrier such as a fatty oil. In addition, the unit dosage form may contain various other substances that alter the physical form of the dosage unit, such as coatings of sugar, shellac, or other enteric solvents.

The compounds may be administered as a component of elixirs, suspensions, syrups, wafers, chewable tablets and the like. In addition to the active compounds, syrups may contain sucrose as a sweetener and certain preservatives, dyes and colorants and flavors.

The compound or pharmaceutically acceptable prodrugs or salts thereof may also be admixed with other active substances which do not impair the desired effect, or with substances which supplement the desired effect, such as antibiotics, antifungals, anti-inflammatory agents or other antiviral compounds. Solutions or suspensions for parenteral, intradermal, subcutaneous, or topical administration may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulphite; chelating agents such as ethylenediamine tetraacetic acid; buffers, such as acetates, citrates or phosphates, and tonicity adjusting agents, such as sodium chloride or dextrose. Parenteral formulations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

If administered intravenously, the preferred carrier is physiological saline or Phosphate Buffered Saline (PBS).

Transdermal preparation

In some embodiments, the composition is in the form of a transdermal formulation, such as a formulation used in FDA approved agonist rotigotine (rotigitine) transdermal patch (Neupro patch). Another suitable formulation is described in U.S. publication No. 20080050424, entitled "Transdermal Therapeutic System for Treating Parkinsonism". The formulation includes a silicone or acrylate based binder and may include an additive that increases the solubility of the active agent in an amount effective to increase the dissolution capacity of the matrix for the active agent.

The transdermal formulation may be a single phase matrix (matrix) comprising a backing layer, a self-adhesive matrix containing the active substance and a protective film to be removed prior to use. More complex embodiments include a multi-layer substrate that may also contain a non-adhesive layer and a control film. If a polyacrylate adhesive is used, it can be crosslinked with polyvalent metal ions (such as zinc, calcium, aluminum or titanium ions), for example aluminum acetylacetonate and titanium acetylacetonate.

When silicone adhesives are used, they are typically polydimethylsiloxanes. However, in principle other organic residues may be present, such as ethyl or phenyl, instead of methyl. Because the active compound is an amine, it may be advantageous to use an amine-resistant binder. A representative amine-resistant adhesive is described, for example, in EP 0 180 377.

Representative acrylate-based polymeric binders include acrylic acid, acrylamide, hexyl acrylate, 2-ethylhexyl acrylate, hydroxyethyl acrylate, octyl acrylate, butyl acrylate, methyl acrylate, glycidyl acrylate, methacrylic acid, methacrylamide, hexyl methacrylate, 2-ethylhexyl methacrylate, octyl methacrylate, methyl methacrylate, glycidyl methacrylate, vinyl acetate, vinyl pyrrolidone, and combinations thereof.

The adhesive must have a dissolving capacity suitable for the active substance and the active substance must be able to move within the matrix and to pass through the contact surface to the skin. Transdermal formulations with appropriate transdermal transport of the active substance can be readily formulated by the person skilled in the art.

Certain pharmaceutically acceptable salts tend to be more preferred for transdermal formulations because they can aid in the barrier of the active substance across the stratum corneum. Examples include fatty acid salts such as stearates and oleates. Oleate and stearate are relatively lipophilic and may even act as penetration enhancers in the skin.

Penetration enhancers may also be used. Representative penetration enhancers include fatty alcohols, fatty acids, fatty acid esters, fatty acid amides, glycerol or fatty acid esters thereof, N-methylpyrrolidone, terpenes (such as limonene, alpha-pinene, alpha-terpineol, carvone, carveol, limonene oxide, pinene oxide, and 1, 8-eucalyptol).

Patches may generally be prepared by dissolving or suspending the active agent in ethanol or another suitable organic solvent, followed by addition of the binder solution with stirring. Additional auxiliary substances may be added to the binder solution, to the active substance solution or to the active substance-containing binder solution. The solution may then be applied to a suitable sheet, the solvent removed, the backing layer laminated to the matrix layer, and the patch punched out of the overall laminate.

Nanoparticle compositions

The compounds described herein may also be administered in the form of nanoparticle compositions. In one embodiment, a controlled release nanoparticle formulation comprises a nanoparticle active agent to be administered and a rate controlling polymer that extends the release of the agent after administration. In this embodiment, the composition may release the active agent for a period of about 2 to about 24 hours or up to 30 days or more after administration. Representative controlled release formulations comprising active agents in nanoparticle form are described, for example, in U.S. patent No. 8,293,277.

Nanoparticle compositions can comprise particles of the active agents described herein having non-crosslinked surface stabilizers adsorbed on or bound to their surfaces.

The average particle size of the nanoparticles is typically less than about 800nm, more typically less than about 600nm, more typically less than about 400nm, less than about 300nm, less than about 250nm, less than about 100nm, or less than about 50nm. In one aspect of this embodiment, at least 50% of the active agent particles have an average particle size of less than about 800, 600, 400, 300, 250, 100, or 50nm, respectively, as measured by light scattering techniques.

A variety of surface stabilizers are commonly used with nanoparticle compositions to prevent agglomeration or aggregation of the particles. Representative surface stabilizers are selected from the group consisting of: gelatin, lecithin, dextran, gum arabic, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, polysilritol (cetomacrogol) emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphate esters, sodium dodecyl sulfate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose phthalate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, tyloxapol (tyloxapol) poloxamers (poloxamers), poloxamers (poloxamines), poloxamines 908, sodium dialkyl sulfosuccinates, sodium lauryl sulfate, alkylaryl polyether sulfonates, mixtures of sucrose stearates and sucrose distearates, p-isononylphenoxypoly- (glycidol), SA9OHCO, decanoyl-N-methylglucamide, N-decyl-D-glucopyranoside, N-decyl-D-maltopyranoside, N-dodecyl-D-maltoside, heptanoyl-N-methylglucamide, N-heptyl-D-glucopyranoside, N-heptyl-D-thioglucoside, N-hexyl-D-glucopyranoside, nonanoyl-N-methylglucamide, N-nonyl-D-glucopyranoside, octanoyl-N-methylglucamide, N-octyl-D-glucopyranoside and octyl-D-thiopyranoside. Lysozyme may also be used as a surface stabilizer for the nanoparticle composition. Certain nanoparticles, such as poly (lactic-co-glycolic acid) (PLGA) -nanoparticles, are known to target the liver when administered Intravenously (IV) or Subcutaneously (SQ).

Representative rate controlling polymers into which nanoparticles may be formulated include chitosan, polyethylene oxide (PEO), polyvinyl acetate phthalate, gum arabic, agar, guar gum, cereal gums, dextran, casein, gelatin, pectin, carrageenan, wax, shellac, hydrogenated vegetable oils, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), sodium carboxymethyl cellulose (CMC), poly (ethylene) oxide, alkyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydrophilic cellulose derivatives, polyethylene glycol, polyvinylpyrrolidone, cellulose acetate butyrate, cellulose acetate phthalate, cellulose acetate trimellitate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetal diethylaminoacetate, poly (alkyl methacrylate), poly (vinyl acetate), polymers derived from and copolymers of acrylic or methacrylic acid and their respective esters and methacrylic acid derived from and their respective copolymers.

Methods of preparing nanoparticle compositions are described, for example, in U.S. Pat. nos. 5,518,187 and 5,862,999, both in regard to "Method of Grinding Pharmaceutical Substances"; U.S. patent No. 5,718,388, for "Continuous Method of Grinding Pharmaceutical Substances"; and U.S. Pat. No. 5,510,118, for "Process of Preparing Therapeutic Compositions Containing Nanoparticles".

Nanoparticle compositions are also described, for example, in U.S. patent No. 5,298,262 with respect to "Use of Ionic Cloud Point Modifiers to Prevent Particle Aggregation During Sterilization"; U.S. patent No. 5,302,401 pertains to "Method to Reduce Particle Size Growth During Lyophilization"; U.S. Pat. No. 5,318,767 pertains to "X-Ray Contrast Compositions Useful in Medical Imaging"; U.S. Pat. No. 5,326,552 pertains to "Novel Formulation For Nanoparticulate X-Ray Blood Pool Contrast Agents Using High Molecular Weight Non-ionic Surfactants"; U.S. Pat. No. 5,328,404 pertains to "Method of X-Ray Imaging Using Iodinated Aromatic Propanedioates"; U.S. patent No. 5,336,507 pertains to "Use of Charged Phospholipids to Reduce Nanoparticle Aggregation"; U.S. Pat. No. 5,340,564 pertains to "Formulations Comprising Olin 10-G to Prevent Particle Aggregation and Increase Stability"; U.S. Pat. No. 5,346,702 pertains to "Use of Non-Ionic Cloud Point Modifiers to Minimize Nanoparticulate Aggregation During Sterilization"; U.S. Pat. No. 5,349,957 pertains to "Preparation and Magnetic Properties of Very Small Magnetic-Dextran Particles"; U.S. patent No. 5,352,459 pertains to "Use of Purified Surface Modifiers to Prevent Particle Aggregation During Sterilization"; U.S. Pat. nos. 5,399,363 and 5,494,683, both for "Surface Modified Anticancer Nanoparticles"; U.S. Pat. No. 5,401,492 pertains to "Water Insoluble Non-Magnetic Manganese Particles as Magnetic Resonance Enhancement Agents"; U.S. Pat. No. 5,429,824 pertains to "Use of Tyloxapol as a Nanoparticulate Stabilizer"; U.S. Pat. No. 5,447,710 pertains to "Method for Making Nanoparticulate X-Ray Blood Pool Contrast Agents Using High Molecular Weight Non-ionic Surfactants"; U.S. Pat. No. 5,451,393 pertains to "X-Ray Contrast Compositions Useful in Medical Imaging"; U.S. Pat. No. 5,466,440 pertains to "Formulations of Oral Gastrointestinal Diagnostic X-Ray Contrast Agents in Combination with Pharmaceutically Acceptable Clays"; U.S. patent No. 5,470,583 pertains to "Method of Preparing Nanoparticle Compositions Containing Charged Phospholipids to Reduce Aggregation"; U.S. Pat. No. 5,472,683 pertains to "Nanoparticulate Diagnostic Mixed Carbamic Anhydrides as X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging"; U.S. Pat. No. 5,500,204 pertains to "Nanoparticulate Diagnostic Dimers as X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging"; U.S. patent No. 5,518,738 pertains to "Nanoparticulate NSAID Formulations"; U.S. Pat. No. 5,521,218 pertains to "Nanoparticulate Iododipamide Derivatives for Use as X-Ray Contrast Agents"; U.S. Pat. No. 5,525,328 pertains to "Nanoparticulate Diagnostic Diatrizoxy Ester X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging"; U.S. Pat. No. 5,543,133 pertains to "Process of Preparing X-Ray Contrast Compositions Containing Nanoparticles"; U.S. patent No. 5,552,160 pertains to "Surface Modified NSAID Nanoparticles"; U.S. patent No. 5,560,931 pertains to "Formulations of Compounds as Nanoparticulate Dispersions in Digestible Oils or Fatty Acids"; U.S. Pat. No. 5,565,188 pertains to "Polyalkylene Block Copolymers as Surface Modifiers for Nanoparticles"; U.S. Pat. No. 5,569,448 pertains to "Sulfated Non-ionic Block Copolymer Surfactant as Stabilizer Coatings for Nanoparticle Compositions"; U.S. patent No. 5,571,536 pertains to "Formulations of Compounds as Nanoparticulate Dispersions in Digestible Oils or Fatty Acids"; U.S. Pat. No. 5,573,749 pertains to "Nanoparticulate Diagnostic Mixed Carboxylic Anhydrides as X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging"; U.S. Pat. No. 5,573,750 pertains to "Diagnostic Imaging X-Ray Contrast Agents"; U.S. patent No. 5,573,783 pertains to "Redispersible Nanoparticulate Film Matrices With Protective Overcoats"; U.S. Pat. No. 5,580,579 relates to "Site-specific Adhesion Within the GI Tract Using Nanoparticles Stabilized by High Molecular Weight, linear Polymers"; U.S. patent No. 5,585,108 pertains to "Formulations of Oral Gastrointestinal Therapeutic Agents in Combination with Pharmaceutically Acceptable Clays"; U.S. Pat. No. 5,587,143 to "butyl Oxide-Ethylene Oxide Block Copolymers Surfactants as Stabilizer Coatings for Nanoparticulate Compositions"; U.S. patent No. 5,591,456 pertains to "Milled Naproxen with Hydroxypropyl Cellulose as Dispersion Stabilizer"; U.S. Pat. No. 5,593,657 pertains to "Novel Barium Salt Formulations Stabilized by Non-ionic and Anionic Stabilizers"; U.S. patent No. 5,622,938 pertains to "Sugar Based Surfactant for Nanocrystals"; U.S. Pat. No. 5,628,981 pertains to "Improved Formulations of Oral Gastrointestinal Diagnostic X-Ray Contrast Agents and Oral Gastrointestinal Therapeutic Agents"; U.S. Pat. No. 5,643,552 pertains to "Nanoparticulate Diagnostic Mixed Carbonic Anhydrides as X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging"; U.S. patent No. 5,718,388 pertains to "Continuous Method of Grinding Pharmaceutical Substances"; U.S. patent No. 5,718,919 pertains to "Nanoparticles Containing the R (-) Enantiomer of Ibuprofen"; U.S. patent No. 5,747,001 pertains to "Aerosols Containing Beclomethasone Nanoparticle Dispersions"; U.S. patent No. 5,834,025 pertains to "Reduction of Intravenously Administered Nanoparticulate Formulation Induced Adverse Physiological Reactions"; U.S. patent No. 6,045,829"Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors Using Cellulosic Surface Stabilizers"; U.S. patent No. 6,068,858 relates to "Methods of Making Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors Using Cellulosic Surface Stabilizers"; U.S. patent No. 6,153,225 pertains to "Injectable Formulations of Nanoparticulate Naproxen"; U.S. patent No. 6,165,506 pertains to "New Solid Dose Form of Nanoparticulate Naproxen"; U.S. patent No. 6,221,400 relates to "Methods of Treating Mammals Using Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors"; U.S. Pat. No. 6,264,922 pertains to "Nebulized Aerosols Containing Nanoparticle Dispersions"; U.S. patent No. 6,267,989 pertains to "Methods for Preventing Crystal Growth and Particle Aggregation in Nanoparticle Compositions"; U.S. Pat. No. 6,270,806 pertains to "Use of PEG-Derivatized Lipids as Surface Stabilizers for Nanoparticulate Compositions"; U.S. patent No. 6,316,029 for "Rapidly Disintegrating Solid Oral Dosage Form", U.S. patent No. 6,375,986 for "Solid Dose Nanoparticulate Compositions Comprising a Synergistic Combination of a Polymeric Surface Stabilizer and Dioctyl Sodium Sulfosuccinate"; U.S. patent No. 6,428,814 pertains to "Bioadhesive nanoparticulate compositions having cationic surface stabilizers"; U.S. patent No. 6,431,478 pertains to "Small Scale Mill"; and U.S. patent No. 6,432,381 to "Methods for targeting drug delivery to the upper and/or lower gastrointestinal tract," all of which are specifically incorporated by reference. Furthermore, in U.S. patent application No. 20020012675A1, published at 1/31 2002, regarding "Controlled Release Nanoparticulate Compositions", nanoparticle compositions are described and specifically incorporated by reference.

Amorphous small particle compositions are described, for example, in U.S. patent No. 4,783,484 for "Particulate Composition and Use Thereof as Antimicrobial Agent"; U.S. Pat. No. 4,826,689 pertains to "Method for Making Uniformly Sized Particles from Water-Insoluble Organic Compounds"; U.S. Pat. No. 4,997,454 pertains to "Method for Making Uniformly-Sized Particles From Insoluble Compounds"; U.S. Pat. No. 5,741,522 pertains to "Ultrasmall, non-aggregated Porous Particles of Uniform Size for Entrapping Gas Bubbles Within and Methods"; and U.S. patent No. 5,776,496, for "Ultrasmall Porous Particles for Enhancing Ultrasound Back Scatter".

Certain nanoformulations can reduce solubility problems by enhancing absorption of the drug by releasing the drug into the intestinal lumen in a controlled manner. The intestinal wall is intended to absorb nutrients and act as a barrier to pathogens and macromolecules. Small amphiphilic and lipophilic molecules can be absorbed by partitioning into lipid bilayers and by passive diffusion through intestinal epithelial cells, whereas the absorption of nano-formulations may be more complex due to the intrinsic properties of the intestinal wall. The first physical barrier to oral absorption of nanoparticles is the mucous barrier covering the intestinal and colonic luminal surfaces. The mucus barrier consists of distinct layers, consisting mainly of highly glycosylated proteins called mucins, which may prevent the absorption of certain nano-formulations. Modifications may be made to give nanoformulations with increased mucus permeability (Ensign et al, "Mucus penetrating nanoparticles: biophysical tool and method of drug and gene delivery," Adv Mater 24:3887-3894 (2012)).

After passing through the mucus layer, transport of the nanoagent across intestinal epithelial cells can be regulated by several steps, including cell surface binding, endocytosis, intracellular transport, and exocytosis, to achieve transcytosis (transcytosis) which may involve multiple subcellular structures. In addition, the nanoformulation can be transported between cells through an open tight junction, which is defined as endocytosis (paracytosis). Non-phagocytic pathways, including clathrin-mediated and pit (cavelae) -mediated endocytosis and megaloblastic, are the most common mechanisms of absorption of nanomaterials by the oral route.

Non-oral administration may provide a variety of benefits, such as direct targeting to the desired site of action and prolonged action of the drug. Transdermal administration has been optimized for nanoformulations, such as Solid Lipid Nanoparticles (SLN) and NE, which are characterized by good biocompatibility, low cytotoxicity and ideal drug release modulating effect (Cappel and Kreuter, "Effect of nanoparticles on transdermal drug release.J Microencapulous 8:369-374 (1991)). Nasal administration of the nanoformulations enables them to penetrate the nasal mucosa via the transmucosal route or via a carrier or receptor mediated transport process by endocytosis (ilm, "Nanoparticulate systems for nasal delivery of drugs: a real improvement over simple systems. Pulmonary administration can provide a large surface area and be relatively easy to access. The mucus barrier, metabolic enzymes in the tracheobronchial region, and macrophages in the alveoli are often the primary barriers to drug permeation. Particle size is a major factor in determining the diffusion of the nanoformulation in the bronchial tree, particles in the nanoscale region are more likely to reach the alveolar region, particles between 1 and 5 μm in diameter are expected to deposit in the bronchioles (Musante et al, "Factors affecting the deposition of inhaled porous drug particles," J Pharm Sci 91:1590-1600 (2002)). For larger particles, absorption has been shown to be limited, possibly due to inability to cross the air-blood barrier. The particles may gradually release the drug so that the drug may permeate into the bloodstream, or the particles may be phagocytosed by alveolar macrophages (Bailey and Berkland, "Nanoparticle formulations in pulmonary drug delivery," med.res.rev.,29:196-212 (2009)).

The penetration of certain nanoformulations through biological membranes at the site of absorption is minimal, for which intravenous administration may be the preferred route to achieve effective distribution in vivo (Wacker, "Nanocarriers for intravenous injection-The long hard road to the market," int.j. Pharm.,457:50-62., 2013).

The distribution of the nanoformulation may vary greatly depending on the delivery system used, the nature of the nanoformulation, the variability between individuals, and the rate of drug loss of the nanoformulation. Certain nanoparticles, such as Solid Drug Nanoparticles (SDN), can improve drug absorption, which does not require them to reach the systemic circulation intact. Other nanoparticles survive the absorption process, thereby altering the distribution and clearance of the contained drug.

A nanofabric of a certain size and composition may diffuse through a well-defined process (e.g., enhanced permeability and retention effects) in a tissue while other nanofabrics accumulate in a specific cell population, which allows one nanofabric to target a specific organ. While complex biological barriers can protect organs from exogenous compounds, the Blood Brain Barrier (BBB) is a barrier to many therapeutic agents. There are many different types of cells in the BBB, including endothelial cells, microglia, pericytes and astrocytes, and the BBB exhibits a very tight junction, as well as a highly active efflux mechanism, limiting penetration of most drugs. Transport across the BBB is generally limited to small lipophilic molecules and nutrients carried by a particular transporter. One of the most important mechanisms regulating the diffusion of nanoformulations into the brain is through endocytosis of brain capillary endothelial cells.

Recent studies have correlated particle properties with the entry route of the nanofabricated formulation and processing in the endothelial barrier of the human BBB, indicating that the permeation of uncoated nanoparticles through the BBB is limited and that surface modification affects the efficiency and mechanism of endocytosis (Lee et al, "Targeting rat anti-mouse transferrin receptor monoclonal antibodies through blood-brain barrier in mouse," j. Pharmacol. Exp. Ter. 292:1048-1052 (2000)). Thus, surface-modified nanoparticles that cross the BBB and deliver one or more compounds described herein are within the scope of the invention.

Macrophages in the liver are the major source of total macrophages in the body. The cumulating (Kupffer) cells in the liver have many receptors for selectively phagocytizing conditioned particles (receptors for complement proteins and receptors for crystallizable portions of IgG fragments). Phagocytosis may provide a mechanism for targeting macrophages and for localized delivery of compounds described herein (i.e., delivery within macrophages).

Nanoparticles attached to polyethylene glycol (PEG) have minimal interactions with receptors, inhibiting phagocytosis by the mononuclear phagocytic system (Bazile et al, "Stealth Me. PEG-PLA nanoparticles avoid uptake by the mononuclear phagocytes system," J. Pharm. Sci.84:493-498 (1995)).

Representative nanoformulations include inorganic nanoparticles, SDN, SLN, NE, liposomes, polymeric nanoparticles, and dendrimers. The compounds described herein may be contained within the nanofabricated formulation or, in some cases, the inorganic nanoparticles and dendrimers, the compounds attached to the surface. Hybrid nanoformulations comprising elements of more than one nanoformulation class may also be used.

SDN is a lipid-free nanoparticle that can improve the oral bioavailability and exposure of poorly water-soluble drugs (Chan, "Nanodrug particles and nanoformulations for drug delivery," adv. Drug. Deliv. Rev.63:405 (2011)). SDN contains drugs and stabilizers, which are produced using a "top-down" (high pressure homogenization and wet milling) or bottom-up (solvent evaporation and precipitation) process.

SLN consists of one or more lipids, an emulsifier, and water that are solid at room temperature. Lipids used include, but are not limited to, triglycerides, partial glycerides, fatty acids, steroids, and waxes. SLNs are most suitable for delivering highly lipophilic drugs.

Droplets smaller than 1000nm dispersed in an immiscible liquid are classified as NE. NE serves as both a carrier for the hydrophobic agent and a carrier for the hydrophilic agent and can be administered orally, transdermally, intravenously, intranasally and intraocularly. For chronic therapies, oral administration may be preferred, NE being effective to enhance the oral bioavailability of small molecules, peptides and proteins.

Polymeric nanoparticles are solid particles, typically about 200-800nm in size, and may include synthetic and/or natural polymers, which may optionally be pegylated to minimize phagocytosis. Polymeric nanoparticles can improve the bioavailability of drugs and other substances compared to traditional formulations. Their clearance rate depends on several factors, including choice of polymer (including polymer size, polymer charge and targeting ligand), positively charged nanoparticles larger than 100nm are cleared primarily by the liver (Alexis et al Factors affecting the clearance and biodistribution of polymeric nanoparticles mol Pharm 5:505-515 (2008)).

Dendrimers are tree-like nanostructured polymers, typically 10-20nm in diameter.

Liposomes are spherical vesicles that include a phospholipid bilayer. A variety of lipids can be utilized to control the degradation level to some extent. In addition to oral administration, liposomes can also be administered in a variety of ways, including intravenous administration (mccoskill et al, 2013), transdermal administration (Pierre and Dos Santos Miranda Costa, 2011), intravitreal administration (Honda et al, 2013), and by pulmonary administration (chattopladhyy, 2013). Liposomes can be combined with synthetic polymers to form lipid-polymer hybrid nanoparticles, extending their ability to target specific sites in the body. The clearance of liposome-entrapped drug depends on the release of the drug and the destruction of the liposome (uptake of the liposome by phagocytic immune cells, aggregation, pH-sensitive breakdown, etc.) (Ishida et al, "Liposome clearance," Biosci Rep 22:197-224 (2002)).

One or more of these nanoparticle formulations may be used to deliver the active agents described herein to macrophages across the blood brain barrier and other locations as appropriate.

Controlled release formulation

In a preferred embodiment, the active compound is prepared with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including but not limited to implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. For example, enteric coated compounds may be used to protect against gastric acid cleavage. Methods of preparing such formulations will be apparent to those skilled in the art. Suitable materials are also commercially available.

Liposomal suspensions (including but not limited to liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (incorporated by reference). For example, a liposome formulation can be prepared by the following method: suitable lipids, such as stearoyl phosphatidylethanolamine, stearoyl phosphatidylcholine, arachidonoyl phosphatidylcholine (arachadoyl phosphatidyl choline), and cholesterol, are dissolved in an inorganic solvent, and then the inorganic solvent is evaporated, leaving a dried lipid film on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The vessel is then rotated by hand to release the lipid material from the sides of the vessel and disperse the lipid aggregates, thereby forming a liposome suspension.

The terminology used to describe the present invention is commonly used and known by those skilled in the art. As used herein, the following abbreviations have the indicated meanings:

DCM dichloromethane

DIPEA N, N-diisopropylethylamine

DME dimethoxyethane

DMF dimethylformamide

DMSO dimethyl sulfoxide

EDCI N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride

EtOAc ethyl acetate

HATU 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxohexafluorophosphate

HOBt hydroxybenzotriazole

MeOH methanol

THF tetrahydrofuran

X-phos 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl

General procedure for the preparation of active Compounds

Methods for easily preparing active compounds are known in the art and result from selective combinations of known methods. The compounds disclosed herein may be prepared as described in detail below or by other methods known to those of skill in the art. It will be understood by those of ordinary skill in the art that variations in detail may be made without departing from the spirit of the invention and the scope of the invention is in no way limited.

The various reaction schemes are summarized below.

Scheme 1 method of synthesis of compound 5.

Scheme 2 alternative synthetic method of intermediate 4.

Scheme 3 methods of synthesis of compounds 8 and 10.

Scheme 4 alternative synthetic methods for compounds 8 and 10.

Scheme 5 method of synthesis of compounds of formula 15.

Scheme 6 synthesis of compounds of formula 16.

Scheme 7 method for the synthesis of compounds of formula 17.

Scheme 8 method of synthesis of compounds of formula 22.

Scheme 9 alternative synthetic methods for compounds of formula 22.

The compounds of formula a can be accomplished by one of ordinary skill in the art using the methods outlined in the following documents: (a) Wang, l.; sullivan, g.m.; hexamer, l.a.; hasvold, l.a.; thaiji, r.; przytulinska, M.; tao, z.f.; li, G; chen, z.; xiao, z.; gu, W.Z,; xue, j.; bui, m.h.; merta, p.; kovar, p.; bouska, j; zhang, h.; park, c.; stewart, k.d.; sham, h.l.; sowin, t.j.; rosenberg, s.h.; lin, N.H.J.Med.chem.,2007,50 (17), 4162-4176; b) Hasvold LA1, wang L, przytenonska M, xiao Z, chen Z, gu WZ, merta PJ, xue J, kovar P, zhang H, park C, sowin TJ, rosenberg SH, lin NH.Bioorg.Med.chem.Lett.2008,18,2311-2315; c) Giannotti, d.; viti, g.; sbraci, p.; pestellini, v.; volterra, g.; borsini, f.; lecci, a.; meli, a.; dapporto, P.; paoli, p.j.med.chem.,1991,34,1356-1362; c) Rami rez-Mart i nez, j.f.; gonz lez-chu vez, r.; guerro-Alba, R.; reyes-Guterrez, P.E.; martI nez, R.; miranda-Morales, M.; espinosa-Luna, r.; gonz lez-Ch vez, M.M.; barajas-Lvopez, C.molecules,2013,18,894-913 and accomplished by general schemes 1-9.

In the schemes described herein, if an intermediate includes a functional group that may interfere, decompose, or otherwise be converted in certain steps, suitable protecting groups may be used to protect such functional groups. After these steps, the protected functional groups (if any) may be deprotected.

Scheme 1 method of synthesis of compound 5.

Compound 5 can be obtained, for example, by the chemical method described in scheme 1. The compound of formula 1 and the appropriately substituted nitroaniline of formula 2 are prepared in the presence of Cu and an inorganic base (e.g., K ₂ CO ₃ 、Na ₂ CO ₃ Or Cs ₂ CO ₃ ) The reaction in the presence of (3) may provide intermediate 3. Reduction of nitro groups in the presence of Hydrogen, e.g. using Pt/C or using SnCl in EtOAc in an alcoholic solvent system ₂ Reduction of the nitro group gives the compounds of formula 4. Compound 4 may be cyclized in the presence of an acid (e.g., HCl or p-toluene sulfonic acid) (scheme B). Alternatively, compound 4 can be treated with, for example, liOH in a mixture of water and THF under basic conditions to give an acid intermediate, which can then be reacted under classical peptide conditions with a coupling agent (e.g., HATU) in an organic base (e.g., et ₃ N) cyclizing it in the presence (route a).

Scheme 2 alternative synthetic method of intermediate 4.

The compounds of formula 4 may also be prepared by reacting a compound of formula 1 with a diamine of formula 6 in Cu and an inorganic base (e.g., K ₂ CO ₃ 、Na ₂ CO ₃ Or Cs ₂ CO ₃ ) Is prepared by reaction in the presence of (a).

Scheme 3 methods of synthesis of compounds 8 and 10.

The compounds of formulae 8 and 10 may be obtained from compounds of formulae 7 or 9 wherein X is a leaving group such as halogen, triflate, mesylate or tosylate by a coupling reaction of an alkyne, alkyl, alkene, organoborane or organotin derivative under classical palladium-catalysed Sonogashira, heck, suzuki or Stille coupling conditions.

Scheme 4 alternative synthetic methods for compounds 8 and 10.

Alternatively, the compounds of formulas 8 and 10 may be prepared by a boronation reaction of a compound of formulas 7 or 9 wherein X is a leaving group such as halogen, triflate, mesylate or tosylate. Intermediates 11 and 12 can then be reacted with an aryl, heteroaryl, alkene, alkyne containing a leaving group (e.g., halogen, triflate, mesylate, or tosylate) under classical palladium-catalyzed Suzuki coupling conditions.

In one embodiment, R ² And R is ³ And combine to form a heterocyclic ring, which may include five to seven membered rings.

Scheme 5 method of synthesis of compounds of formula 15.

The compounds of formula 15 can be prepared from esters of the general formula prepared by the above chemical methods by treatment with, for example, liOH in a mixture of water and THF under basic conditions to give acid intermediates, which can then be reacted under classical peptide conditions with a coupling agent (e.g., HATU) in an organic base (e.g., et ₃ N) with an amine in the presence of a coupling agent.

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Scheme 6 synthesis of compounds of formula 16.

The compounds of formula 16 may be obtained by treatment with an amination agent such as O- (2, 4-dinitrophenyl) hydroxylamine in the presence of a base.

Scheme 7 method for the synthesis of compounds of formula 17.

The compounds of formula 17 may be obtained by treatment with an oxidizing agent, such as mCPBA.

Scheme 8 method of synthesis of compounds of formula 22.

The compounds of formula 22 can be obtained by the chemical methods described in scheme 8. Reaction of a compound of formula 18 with an appropriately substituted aniline of formula 19 in the presence of an organic base (e.g., pyridine or trimethylamine) can provide intermediate 20. Reduction of nitro groups in the presence of Hydrogen, e.g. using Pt/C or using SnCl in EtOAc in an alcoholic solvent system ₂ Reduction of the nitro group gives compounds of formula 21. Compound 21 may be formed between Cu and an inorganic base (e.g., K ₂ CO ₃ 、Na ₂ CO ₃ Or Cs ₂ CO ₃ ) Cyclization is carried out in the presence of a catalyst.

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Scheme 9 alternative synthetic methods for compounds of formula 22.

Alternatively, the compound of formula 22 may be obtained by a chemical reaction as described in scheme 9. Reaction of a compound of formula 18 with an appropriately substituted aniline of formula 23 in the presence of an organic base (e.g., pyridine or triethylamine) can provide intermediate 24. Reduction of nitro groups in the presence of Hydrogen, e.g. using Pt/C or using SnCl in EtOAc in an alcoholic solvent system ₂ Reduction of the nitro group gives compounds of formula 25. NaNO can be used for example ₂ 、CF ₃ COOH and NaN ₃ An azide of 25 is performed to give the compound of formula 26. Can be prepared in a high boiling point solvent (e.g. dihexylether) Cyclizing compound 26 at an elevated temperature.

Substitution reaction of aromatic rings

In the various reaction schemes shown above, the aromatic ring is surrounded by various R ¹ And R is ² And (3) substituent groups are substituted. It is known in the art how to provide substituents on aromatic rings. For example, where it is desired to provide substitution on one or both aromatic rings, electrophilic aromatic substitution may be used to provide the desired functionality. For example, alkyl, aryl, heteroaryl, alkylaryl, arylalkyl, alkenyl, alkynyl, and acyl groups can be added using Friedel-Crafts alkylation/arylation/acylation reactions. Other electrophilic aromatic substitution reactions may be used, for example to provide halogens, for example by forming chloronium or bromonium ions in situ and reacting them with aromatic rings, or by forming sulfonium or nitronium ions to provide sulfonyl or nitro groups.

Friedel Crafts alkylation reactions are carried out using the appropriate haloalkyl moiety and Lewis acid. The alkyl moiety forms a carbocation, and an electron from the aryl ring forms a bond with the carbocation, thereby positively charging the aryl ring. Then, the aryl ring loses proton. In this way alkyl and alkylaryl moieties (e.g., benzyl moieties) can be added.

The acylation of Friedel Crafts is similar, but an acyl halide (e.g., acyl chloride) is used to place the ketone moiety on the ring. The acid halide may be an alkyl acid (e.g., acetic acid, propionic acid, butyric acid, etc.) or may be an aromatic acid (e.g., benzoic acid, p-toluic acid, etc.).

Friedel Crafts arylation reactions (also known as Scholl reactions) are Lewis acid catalyzed coupling reactions with two aryl rings. Protons lost during the coupling reaction act as additional catalysts. Typical reagents are iron (III) chloride in methylene chloride, copper (II) chloride in methylene chloride, PIFA and boron trifluoride etherate, molybdenum (V) chloride and lead tetraacetate and BF in acetonitrile ₃ 。

Substitution typically occurs at the ortho or para position of the amine group and at the meta position of the nitro group. Thus, depending on the desired functionality and position, it may be desirable to start with an amine group and place substituents. Thus, positions 3, 6 and 8 are typically functionalized using this chemical reaction. Substitution of the naphthalene ring in the meta (i.e., positions 2 and 7) position of the amine group can be performed by oxidizing the amine group to a nitro group, resulting in meta substitution. The nitro group may then be reduced back to an amine group.

Scheme 10 methods of synthesizing compounds of formulas 72, 73 and 74.

Compounds of formulae 72, 73 and 74 can be obtained by chemical reactions described in scheme 10. Phosphate intermediates 69, 70 and 71 can be prepared from 68 by reacting with halogen methyl phosphate in a base (such as, but not limited to, naH or Cs ₂ CO ₃ ) Is formed by reaction in the presence of (a). Alternatively, phosphate intermediates 69, 70 and 71 may be reacted with chloroiodomethane via 68 in a base such as, but not limited to, naH or Cs ₂ CO ₃ Is prepared by reaction in the presence of a phosphate diester salt, wherein the salt may be, but is not limited to Na ⁺ 、K ⁺ Or tetraalkylammonium. Phosphate intermediates 69, 70 and 71 can also be prepared by first reacting 68 with (halomethyl) (4-chlorophenyl) sulfane, then with chlorine, and then substituting the resulting N-chloromethyl intermediate with a phosphate diester salt. Finally, phosphates 69, 70 and 71 are R ₃ When tBu, deprotection under acidic conditions (e.g., but not limited to HCl in dioxane, TFA in toluene, acOH in water and acetonitrile), or when R ₃ When=bn, deprotection can be achieved by hydrogenation.

Deuterium incorporation:

single or multiple replacement of hydrogen with deuterium (carbon-hydrogen bond to carbon-deuterium bond) at the metabolic site of the ROR modulator is expected to slow down the metabolic rate. This may provide a relatively long half-life, as well as a slower rate of clearance from the body. The slow metabolism of therapeutic ROR modulators is expected to add additional advantages to the therapeutic candidate, while other physical or biochemical properties are not affected.

Methods of incorporating deuterium into organic derivatives are well known to those skilled in the art. Representative methods are disclosed in Angew.chem.int.ed.Engl.2007,46,7744-7765. Thus, using these techniques, one or more deuterium atoms may be provided in the ROR modulators described herein.

The invention will be better understood with reference to the following non-limiting examples.

Example 1

Synthesis of Compound 1 and Compounds 34-46.

4-fluoro-3-nitrobenzoic acid methyl ester (28)

4-fluoro-3-nitrobenzoic acid 27 (10 g,54 mmol) was dissolved in methanol (200 mL) and concentrated H at room temperature ₂ SO ₄ (1 mL). The reaction mixture was stirred at 80 ℃ overnight. After the reaction was completed, the solvent was evaporated under reduced pressure. The crude mixture was taken up in H ₂ O (200 ml) was diluted and treated with NaHCO ₃ Alkalizing the saturated solution. The precipitated solid was filtered, washed with water (2 x 100 ml) and dried under vacuum to give compound 28 as a white solid (10.75 g, 84%); ¹ H NMR(400MHz,DMSO-d ₆ ):δ8.54(dd,J＝7.3,2.3Hz,1H),8.31(ddd,J＝8.8,4.3,2.3Hz,1H),7.73(dd,J＝11.1,8.7Hz,1H),3.90(s,3H)； ¹³ C NMR(101MHz,DMSO-d ₆ ):158.7,156.0,136.9,136.8(d,J＝10.8Hz),127.1(d,J＝1.6Hz),126.7(d,J＝3.9Hz),119.4(d,J＝21.7Hz),52.9； ¹⁹ F NMR(377MHz,DMSO)δ-111.97。

4- ((2- (methoxycarbonyl) phenyl) amino) -3-nitrobenzoic acid methyl ester (30)

To a solution of methyl 4-fluoro-3-nitrobenzoate 28 (1 g,6.61 mmol) in NMP (20 mL) was added DIPEA (0.76 mL,19.83 mmol) and methyl 4-fluoro-3-nitrobenzoate 29 (1.5 g,9.92 mmol) at room temperature under an inert atmosphere. The mixture was stirred at 120℃for 14h, after the reaction was completed, the mixture was cooled to room temperature, diluted with diethyl ether (20 ml) and stirred for 1h. The resulting solid was filtered, washed with EtOAc (20 mL) and dried under vacuum to give compound 30 as yellow Color solids (996 mg, 45%); ¹ H NMR(400MHz,DMSO-d ₆ ):δ11.15(s,1H),8.66(d,J＝2.1Hz,1H),8.09–7.96(m,2H),7.71–7.61(m,2H),7.59(d,J＝9.0Hz,1H),7.29(ddd,J＝8.2,5.9,2.1Hz,1H),3.87(s,6H)； ¹³ C NMR(101MHz,DMSO):δ166.6,164.5,142.7,139.7,135.4,134.7,133.9,131.5,128.0,124.2,121.9,120.4,120.0,117.8,52.5,52.3。

3-amino-4- ((2- (methoxycarbonyl) phenyl) amino) -benzoic acid methyl ester (31)

Methyl 4- ((2- (methoxycarbonyl) phenyl) amino) -3-nitrobenzoate 30 (2.5 g,7.5 mmol) and 10% Pd/C (1.25 g,50% wet) in MeOH were stirred at room temperature under a hydrogen atmosphere for 16 hours. After completion of the reaction, the mixture was filtered through celite and washed with 20% MeOH/DCM (250 mL). The filtrate was concentrated and the crude residue was purified by flash column chromatography (AcOEt/hexane 3/7) to give compound 31 as a yellow solid (1.4 g, 62%); ¹ H NMR(400MHz,DMSO-d ₆ )δ8.93(s,1H),7.91(d,J＝8.4Hz,1H),7.52–7.36(m,2H),7.21(s,2H),6.95(d,J＝8.4Hz,1H),6.88–6.72(m,1H),5.19(s,2H),3.85(s,3H),3.80(s.3H)； ¹³ C NMR(101MHz,DMSO)δ167.9,166.3,146.7,142.2,134.3,131.2,130.6,125.5,121.9,118.1,117.7,116.2,114.9,112.3,51.9,51.8。

3-amino-4- ((2-carboxyphenyl) amino) benzoic acid (32)

To methyl 3-amino-4- (((2- (methoxycarbonyl) phenyl) amino) benzoate 31 (1.4 g,4.66 mmol) in THF: H at room temperature ₂ To a solution in a mixture of O (2.5/1, 105 ml) was added lithium hydroxide monohydrate (1.75 g,41.9 mmol). The reaction mixture was stirred at 65 ℃ for 5 hours, followed by removal of volatiles under vacuum. The pH of the residue was acidified to 4 with 2N HCl. The precipitated solid was filtered, washed with water (10 mL) and dried under vacuum to give compound 32 as a white solid (1 g, 80%). ¹ H NMR(400MHz,DMSO-d ₆ )δ12.64(s,2H),9.20(s,1H),7.89(d,J＝7.9Hz,1H),7.44–7.31(m,2H),7.18(s,2H),6.91(d,J＝8.5Hz,1H),6.76(t,J＝7.5Hz,1H),5.06(s,2H)； ¹³ C NMR(101MHz,DMSO)δ169.8,167.4,147.3,142.1,134.0,131.7,130.4,126.6,121.9,118.4,117.3,116.5,114.5,112.8。

11-oxo-10, 11-dihydro-5H-dibenzo [ b, e ] [1,4] diazepine-8-carboxylic acid (33)

A solution of compound 32 (1 g,3.67 mmol) and CDI (2.39 g,14.6 mmol) in THF (40 mL) was stirred at room temperature under an inert atmosphere for 24h. After the reaction was completed, volatiles were removed under vacuum. The pH of the residue was adjusted to 2 with 2N HCl. The precipitated solid was filtered, washed with pentane (10 mL) and dried under vacuum to give compound 33 as a pale green solid (746 g, 80%). ¹ H NMR(400MHz,DMSO-d ₆ )12.66(s,1H),9.93(s,1H),8.28(s,1H),7.70(dd,J＝7.9,1.7Hz,1H),7.57–7.49(m,2H),7.36(td,J＝7.9,1.7Hz,1H),7.02(dd,J＝17.0,8.1Hz,2H),6.91(t,J＝7.4Hz,1H)； ¹³ C NMR(101MHz,DMSO)δ167.4,166.7,148.8,143.8,133.5,132.3,129.2,126.0,125.0,122.5,122.2,121.1,119.4,119.2,SM(IS):m/z:255.4[M+1]。

General procedure I

EDCI, HCl (121 mg,0.629 mmol), HOBt (85 mg,6.29 mmol), amine (0.511 mmol) and DIPEA (205 ml,0.117 mmol) were added to a solution of compound 33 (100 mg,0.393 mmol) in DMF (5 ml) under an inert gas at 0deg.C. The reaction mixture was then stirred at room temperature for 16-24h. After the reaction was completed, water was added. The crude solid was filtered and washed with water. The crude residue was purified by silica gel chromatography (DCM/methanol) to give the desired compound.

8- (4-Benzylpiperidine-1-carbonyl) -10,11 a-dihydro-4 aH-dibenzo [ b, e ] [1,4] diazepin-11 (5H) -one (Compound 1)

Compound 1 was prepared according to general procedure I from 4-benzylpiperidine (91 ml,0.511 mmol). Column chromatography: DCM/MeOH (95:5); pale yellow solid (111 mg, 68%); ¹ H NMR(400MHz,DMSO-d ₆ )δ9.91(s,1H),8.06(s,1H),7.68(d,J＝7.8Hz,1H),7.34(t,J＝7.8Hz,1H),7.27(t,J＝7.8Hz,2H),7.16(d,J＝7.8Hz,3H),7.05-6.99(m,4H),6.89(d,J＝7.8Hz,1H),4.34(s,1H),3.67(s,1H),2.78(s,2H),2.52(s,2H),1.75(s,1H),1.56(s,2H),1.17–1.02(m,2H)； ¹³ C NMR(101MHz,DMSO-d ₆ )δ168.2,167.6,149.6,140.7,140.0,133.4,132.2,130.6,129.3,129.0,128.1,125.8,123.4,122.5,120.9,120.0,119.4,119.1,42.1,37.5,31.6；SM(IS):412.5m/z:[M+1]the method comprises the steps of carrying out a first treatment on the surface of the For C ₂₆ H ₂₆ N ₃ O ₂ HRMS (ESI) [ m+h ]] ⁺ Calculated 412.1947, found 412.2018.

8- (4-phenylpiperazine-1-carbonyl) -5H-dibenzo [ b, e ] [1,4] diazepin-11 (10H) -one (34)

Compound 34 was prepared according to general procedure I from 4-phenylpiperazine (77 ml,0.511 mmol). Column chromatography: DCM/MeOH (95:5); beige solid (100 mg, 64%); ¹ H NMR(400MHz,DMSO-d ₆ )δ9.94(s,1H),8.12(s,1H),7.70(d,J＝7.7Hz,1H),7.37(t,J＝7.6Hz,1H),7.23(t,J＝7.9Hz,2H),7.06(d,J＝2.6Hz,3H),7.01(d,J＝8.0Hz,1H),6.97-6.93(m,3H),6.82(t,J＝7.2Hz,1H),3.72–3.52(m,4H),3.15(s,4H)； ¹³ CNMR(101MHz,DMSO-d ₆ )δ168.4,167.6,150.8,149.5,141.0,133.4,132.2,129.8,129.4,129.0,123.8,122.4,120.9,120.4,119.4,119.4,119.1,115.9,48.2；SM(IS):399.5m/z:[M+1]；C ₂₄ H ₂₃ N ₄ O ₂ HRMS (ESI) [ m+h ]] ⁺ Calculated values: 399.1810, found 399.1812

N-nonyl-11-oxo-10, 11-dihydro-5H-dibenzo [ b, e ] [1,4] diazepine-8-carboxamide (35)

Compound 35 is prepared according to general procedure I from nonylamine (93. Mu.l, 0.511 mmol). The reaction mixture was quenched with water and extracted with ethyl acetate (3×5 mL). The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure, and finally purified by column chromatography eluting with DCM/methanol (99:1 to 95/5); yellow solid (70 mg, 47%); ¹ H NMR(400MHz,DMSO-d ₆ )δ9.91(s,1H),8.24(t,J＝5.4Hz,1H),8.13(s,1H),7.69(dd,J＝7.9,1.6Hz,1H),7.47–7.39(m,2H),7.39–7.32(m,1H),7.00(dd,J＝7.9,5.4Hz,2H),6.91(t,J＝7.9Hz,1H),3.20(q,J＝6.6Hz,2H),1.49(d,J＝8.0Hz,2H),1.30–1.22(m,12H),0.85(t,J＝6.6Hz,3H)； ¹³ C NMR(101MHz,DMSO-d ₆ )δ167.5,165.3,149.4,142.4,133.4,132.2,129.5,129.2,123.2,122.4,121.0,120.9,119.1,119.0,31.3,29.1,29.0,28.8,28.7,26.5,22.1,14.0；SM(IS):380.2m/z:[M+1]；C ₂₃ H ₃₀ N ₃ O ₂ HRMS (ESI) [ m+h ]] ⁺ Calculated values: 380.2327, found: 380.2332.

8- (4-Benzylpiperazine-1-carbonyl) -5H-dibenzo [ b, e ] [1,4] diazepin-11 (10H) -one (36)

Compound 36 was prepared according to general procedure I from 1-benzylpiperazine (89 ml,0.511 mmol). Column chromatography: DCM/MeOH (99:1 to 95/5); yellow solid (42 mg, 26%); ¹ H NMR(400MHz,DMSO-d ₆ )9.92(s,1H),8.09(s,1H),7.69(dd,J＝7.9,1.7Hz,1H),7.40–7.27(m,5H),7.25(td,J＝5.9,2.5Hz,1H),7.08–6.93(m,4H),6.91(t,J＝7.9Hz,1H),3.50(s,4H),3.36(s,2H),2.36(s,4H)； ¹³ C NMR(101MHz,DMSO-d ₆ )168.3,167.6,149.5,141.0,137.8,133.4,132.2,130.0,129.3,128.9,128.2,127.0,123.7,122.4,120.9,120.3,119.4,119.1,61.9,52.6；SM(IS):413.5m/z:[M+1]；C ₂₅ H ₂₅ N ₄ O ₂ HRMS (ESI) [ m+h ]] ⁺ Calculated values: 413.1899, found: 413.1970.

8- (4- (4-Fluorobenzyl) piperidine-1-carbonyl) -5H-dibenzo [ b, e ] [1,4] diazepin-11 (10H) -one (37)

Compound 37 was prepared according to general procedure I from 4- (4-fluorobenzyl) piperidine (99 mg,0.511 mmol). Column chromatography: DCM: meOH (99/1 to 95/5); yellow solid (74 mg, 43%); ¹ H NMR(400MHz,DMSO-d ₆ )δ9.92(s,1H),8.07(s,1H),7.69(dd,J＝8.0,1.7Hz,1H),7.36(ddd,J＝8.0,7.2,1.7Hz,1H),7.24–7.19(m,2H),7.13–7.07(m,2H),7.04–6.95(m,4H),6.91(ddd,J＝8.0,7.2,1.7Hz,1H),4.36(s,1H),3.68(s,1H),2.86(s,2H),2.52(s,2H),1.75(s,1H),1.56(s,2H),1.11(qd,J＝12.3,4.2Hz,2H). ¹³ C NMR(101MHz,DMSO-d ₆ ):δ133.4,132.2,130.7(d,J＝7.8Hz),123.4,120.9,120.0,119.3,119.1,114.8(d,J＝20.9Hz),41.1,37.5. ¹⁹ F NMR(377MHz,DMSO-d ₆ ):δ-117.51(s).SM(IS):430.5m/z:[M+1]；C ₂₆ H ₂₅ FN ₃ O ₂ HRMS (ESI) [ m+h ]] ⁺ Calculated values: 430.1853, found: 430.1926.

8- (4-morpholine-4-carbonyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (38)

Compound 38 was prepared according to general procedure I from morpholine (44 ml,0.511 mmol). After 16 hours, the reaction was quenched with water and extracted with ethyl acetate (3×5 mL). The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure, purified by column chromatography, eluting with DCM/methanol (99:1 to 95/5); yellow solid (54 mg, 43%); ¹ H NMR(400MHz,DMSO-d ₆ )δ9.92(s,1H),8.10(s,1H),7.70(dd,J＝7.9,1.6Hz,1H),7.36(ddd,J＝8.6,7.9,1.7Hz,1H),7.06–6.94(m,4H),6.96–6.87(m,1H),3.58(d,J＝5.0Hz,4H),3.47(s,4H)； ¹³ C NMR(101MHz,DMSO-d ₆ ):δ168.5,167.6,149.5,141.1,133.4,132.2,129.6,129.4,123.8,122.4,120.9,120.5 119.4,119.1,66.2,40.1.SM(IS):324.5m/z:[M+1]；C ₁₈ H ₁₈ N ₃ HRMS (ESI) of O [ m+h] ⁺ Calculated values: 324.1270, found: 324.1341

N- (3- (1H-imidazol-1-yl) propyl) -11-oxo-10, 11-dihydro-5H-dibenzo [ b, e ] [1,4] diazepine-8-carboxamide (39)

Compound 39 was prepared according to general procedure I from 3- (1H-imidazol-1-yl) propan-1-amine (0.511 mmol). After 16h, 30ml of water are added and the mixture is extracted twice with 20ml of ethyl acetate. The combined organic layers were dried over MgSO ₄ Dried, filtered and evaporated under vacuum. The crude solid was purified by column chromatography eluting with DCM/methanol (99:1 to 95/5); yellow solid (40 mg, 28%). ¹ H NMR(400MHz,DMSO-d ₆ )δ9.92(s,1H),8.34(t,J＝5.6Hz,1H),8.15(s,1H),7.70(dd,J＝7.9,1.7Hz,1H),7.65(d,J＝1.3Hz,1H),7.53–7.41(m,2H),7.36(ddd,J＝8.7,7.2,1.7Hz,1H),7.21(d,J＝1.3Hz,1H),7.08–6.97(m,2H),6.93–6.87(m,2H),4.00(t,J＝6.9Hz,2H),3.19(q,J＝6.9Hz,2H),1.93(p,J＝6.9Hz,2H), ¹³ C NMR(101MHz,DMSO-d ₆ ):δ167.5,165.7,149.3,142.5,137.3,133.4,132.2,129.3,129.2,128.4,123.3,122.4,121.0,121.0,119.3,119.1,119.0,43.7,36.4,30.8.SM(IS):362.4m/z:[M+1]；C ₂₀ H ₂₀ N ₅ O ₂ HRMS(ESI)[M+H] ⁺ Calculated values: 362.1539, found: 362.1609

8- (4- (2-Fluorobenzyl) piperidine-1-carbonyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (40)

Compound 40 was prepared according to general procedure I from 4- (2-fluorobenzyl) piperidine (99 mg,0.511 mmol). Column chromatography: DCM: meOH (99/1 to 95/5); yellow solid (135 mg, 80%); ¹ H NMR(400MHz,DMSO-d ₆ ):δ9.92(s,1H),8.08(s,1H),7.70(d,J＝7.8Hz,1H),7.36(t,J＝7.6Hz,1H),7.27(d,J＝8.6Hz,2H),7.14(q,J＝7.3Hz,2H),7.00(t,J＝5.9Hz,4H),6.92(t,J＝7.4Hz,1H),4.35(s,1H),3.69(s,1H),2.75(s,2H),2.58(d,J＝7.0Hz,2H),1.79(s,1H),1.58(s,2H),1.15(q,J＝12.5Hz,2H)； ¹³ C NMR(101MHz,DMSO-d ₆ )δ168.3,167.6,161.8,159.4,149.6,140.8,133.4,132.2,131.7(d,J＝5.1Hz),130.6,129.3,128.1(d,J＝8.3Hz),126.6(d,J＝15.9Hz),124.2(d,J＝3.3Hz),123.4,122.5,120.9,120.0,119.4,119.1,115.1(d,J＝22.3Hz),36.6,35.0,31.7. ¹⁹ F NMR(377MHz,DMSO-d ₆ )δ-118.26.,SM(IS):430.1m/z:[M+1]；C ₂₆ H ₂₄ FN ₃ O ₂ HRMS (ESI) [ m+h ]] ⁺ Calculated values: 430.1853, found: 430.1927.

8- (piperidine-1-carbonyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (41)

Compound 41 was prepared according to general procedure I from piperidine (58 ml,0.511 mmol). After 24h, 30ml of water are added and the mixture is extracted twice with 20ml of ethyl acetate. The combined organic layers were dried over MgSO ₄ Dried, filtered and concentrated under vacuum. The crude solid was purified by column chromatography eluting with DCM/methanol (99:1 to 95/5); yellow solid (20 mg, 16%). ¹ H NMR(400MHz,DMSO-d ₆ )δ9.92(s,1H),8.07(s,1H),7.69(dd,J＝7.9,1.7Hz,1H),7.40–7.33(m,1H),7.05–6.96(m,4H),6.95–6.88(m,1H),3.42 -3.31(m,4H),1.60(q,J＝5.6Hz,2H),1.54–1.41(m,4H). ¹³ C NMR(101MHz,DMSO)δ168.2,167.7,149.6,140.7,133.4,132.2,130.7,129.4,123.4,122.5,120.9,120.0,119.4,119.1,25.7,24.7；C ₁₉ H ₂₀ FN ₃ O ₂ HRMS (ESI) [ m+h ]] ⁺ Calculated values: 322.1477, found: 322.1548.

8- (4-Benzylpiperidine-1-carbonyl) -2-bromo-5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (42)

Compound 42 was prepared according to general procedure I from 4-phenylpiperidine (82 mg,0.511 mmol). Column chromatography: DCM: meOH (99/1 to 95/5); yellow solid (67 mg, 43%); ¹ H NMR(400MHz,DMSO-d ₆ )δ9.94(s,1H),8.09(s,1H),7.70(dd,J＝7.9,1.7Hz,1H),7.38–7.32(m,1H),7.32–7.26(m,4H),7.21(d,J＝7.0Hz,1H),7.08–7.02(m,3H),7.00(dd,J＝8.1,1.1Hz,1H),6.94–6.88(m,1H),4.71–4.39(m,1H),3.95–3.66(m,1H),3.11–2.98(s,1H),2.80(t,J＝12.0Hz,2H),1.98–1.73(m,2H),1.61(td,J＝12.6,4.1Hz,2H).SM(IS):398.2m/z:[M+1]。

8- (4-phenethylpiperidine-1-carbonyl) 5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (43)

Compound 43 was prepared according to general procedure I from 4-phenethyl piperidine (96 mg,0.511 mmol). Column chromatography: DCM/methanol (99/1 to 95/5); yellow solid (67 mg, 40%); ¹ H NMR(400MHz,DMSO-d ₆ 9.92(s,1H),8.07(s,1H),7.69(dd,J＝7.9,1.7Hz,1H),7.36(ddd,J＝8.5,7.9,1.7Hz,1H),7.28(t,J＝7.9Hz,2H),7.23–7.14(m,3H),7.03-6.97(d,J＝7.0Hz,4H),6.95–6.89(m,1H),4.38(s,1H),3.69(s,1H),2.90(s,2H),2.60(t,J＝7.5Hz,3H),1.73(s,2H),1.52(d,J＝7.4Hz,3H),1.22–0.99(m,H). ¹³ C NMR(101MHz,DMSO-d ₆ )168.6,168.11,150.0,142.7,141.2,133.9,132.6,131.1,129.8,128.7,128.7,126.1,123.8,122.9,121.4,120.5,119.8,119.6,4,38.3,35.4,32.6.SM(IS):426.2m/z:[M+1]。

n- (1-Benzylpiperidin-4-yl) -11-oxo-10, 11-dihydro-5H-dibenzo [ b, e ] [1,4] diazepine-8-carboxamide (44)

Compound 44 was prepared according to general procedure I from 1-benzyl-4-aminopiperidine (104 ml,0.511 mmol). Column chromatography: DCM/methanol (99/1 to 95/5); yellow solid (60 mg, 35%); ¹ H NMR(400MHz,DMSO-d ₆ )9.89(s,1H),8.14(s,1H),8.06(d,J＝7.6Hz,1H),7.69(dd,J＝7.9,1.6Hz,1H),7.45(d,J＝7.0Hz,2H),7.39–7.28(m,5H),7.27-7.23(m,1H),7.00(t,J＝7.9Hz,1H),6.95–6.85(m,1H),3.73(d,J＝7.0Hz,1H),3.46(s,2H),2.81(d,J＝9.8Hz,2H),2.01(s,2H),1.75(d,J＝12.4Hz,2H),1.56(td,J＝11.8,3.6Hz,2H)； ¹³ C NMR(101MHz,DMSO-d ₆ ):168.0,165.3,149.8,142.9,139.1,133.8,132.7,129.9,129.6,129.2,128.6,127.3,123.9,122.8,121.5,121.4,119.6,119.4,62.6,52.7,47.3,32.0.SM(IS):427.3m/z:[M+1]。

8- (4-Benzoylpiperidine-1-carbonyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (45)

Compound 45 was prepared according to general procedure I from 4-benzoylpiperidine (89 ml,0.511 mmol). Column chromatography: DCM/methanol (99/1 to 95/5); yellow solid (114 mg, 68%); ¹ H NMR(400MHz,DMSO-d ₆ )9.93(s,1H),8.09(s,1H),8.01(d,J＝7.7Hz,2H),7.74–7.61(m,2H),7.55(t,J＝7.5Hz,2H),7.42–7.31(m,1H),7.02(d,J＝7.4Hz,4H),6.91(t,J＝7.5Hz,1H),4.40(s,1H),3.86–3.65(m,1H),3.08(s,4H),1.82(s,2H),1.52(d,J＝13.8Hz,3H), ¹³ C NMR(101MHz,DMSO-d ₆ ):δ201.7,168.4,167.6,149.5,140.8,135.4,133.4,133.26,132.2,130.3,129.3,128.8,128.2,123.5,122.4,120.9,120.0,119.4,119.1,42.4,28.5；SM(IS):426.2m/z:[M+1]。

8- (4- (3-Fluorobenzyl) piperidine-1-carbonyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (46)

Compound 46 was prepared according to general procedure I using 4- (3-fluorobenzyl) piperidine hydrochloride (115 mg,0.511 mmol). Column chromatography: DCM: meOH (99/1 to 95/5); yellow solid (90 mg, 53%); ¹ H NMR(400MHz,DMSO-d ₆ )δ9.92(s,1H),8.07(s,1H),7.69(dd,J＝7.9,1.7Hz,1H),7.38–7.27(m,2H),7.03–6.96(m,7H),6.93–6.88(m,1H),4.35(s,1H),3.73(s,1H),2.78(s,2H),2.54(d,J＝7.1Hz,2H),1.87(s,1H),1.55(s,2H),1.11(qd,J＝12.1,4.1Hz,2H), ¹⁹ F NMR(377MHz,DMSO-d ₆ )δ-113.89(s),SM(IS):430.1m/z:[M+1]。

scheme 11 synthesis of compounds 47-49.

8- (4-Benzylpiperidine-1-carbonyl) -10-bromo-5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (47)

Compound 1 (100 mg,0.243 mmol) was dissolved in 1ml DMF and then in the chamberAdding Cs at a low temperature ₂ CO ₃ (158 mg, 0.4816 mg) followed by MeI (16 ml,0.267 mmol) was added. The reaction mixture was stirred at room temperature overnight and then with H ₂ O (20 ml) was diluted. The solid obtained is filtered off, with 5ml of H ₂ O was washed and then purified by column chromatography on silica gel (DCM/methanol (99:1)) to give compound 47 as a white solid (80 mg, 78%). ¹ H NMR(400MHz,DMSO-d ₆ )δ8.13(s,1H),7.67(dd,J＝7.9,1.7Hz,1H),7.40–7.33(m,1H),7.32–7.25(m,3H),7.20–7.12(m,4H),7.12–7.06(m,2H),7.00–6.95(m,1H),4.50–4.27(m,1H),3.80–3.56(m,1H),3.61(s,3H),3.10–2.82(m,1H),2.72-2.70(m,1H),2.53(s,2H),1.80–1.74(m,1H),1.67–1.47(m,2H),1.25–1.02(m,2H)； ¹³ C NMR(101MHz,DMSO)δ168.1,167.5,151.2,144.8,140.0,134.6,132.6,132.3,131.5,129.0,128.2,125.8,124.3,123.8,122.0,121.5,119.9,118.8,42.1,40.15,37.7,37.5,31.5.SM(IS):426.2m/z:[M+1]；C ₂₇ H ₂₈ N ₃ O ₂ HRMS(ESI)[M+H] ⁺ Calculated values: 426.2103, found: 426.2176.

8- (4-Benzylpiperidine-1-carbonyl) -10-butyl-5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (48)

Compound 1 (40 mg,0.097 mmol) was dissolved in 0.5ml of DMF, then NaH (5 mg,0.194 mg) was added at room temperature followed by BuI (12 ml,0.106 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was treated with H ₂ O (20 ml) was diluted. The solid obtained is filtered off, with 5ml of H ₂ O-washing and purification by column chromatography on silica gel (DCM/methanol (99:1)) gave compound 48 as a white solid (22.5 mg, 50%); ¹ H NMR(400MHz,DMSO-d ₆ )δ8.09(s,1H),7.68(dd,J＝7.8,1.7Hz,1H),7.43–7.37(m,2H),7.37–7.31(m,2H),7.24(ddd,J＝8.3,5.9,1.9Hz,4H),7.14(ddd,J＝16.2,8.2,1.5Hz,2H),7.03(ddd,J＝8.1,7.3,1.2Hz,1H),4.57–4.36(m,1H),4.08(s,2H),3.80–3.56(m,1H)3.10–2.86(m,2H),2.59(s,2H),1.88–1.77(m,1H),1.72–1.55(m,2H),1.50(dt,J＝14.2,6.8Hz,2H),1.32(dq,J＝14.2,7.3Hz,2H),1.26–1.15(m,2H),0.86(t,J＝7.3Hz,3H).SM(IS):468.1m/z:[M+1]。

10-benzyl-8- (4-benzylpiperidine-1-carbonyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (49)

Compound 1 (40 mg,0.097 mmol) was dissolved in 0.5ml DMF and Cs was then added at room temperature ₂ CO ₃ (63 mg,0.191 mmol) followed by benzyl bromide (13 ml,0.106 mmol). The reaction mixture was stirred at room temperature overnight and then with H ₂ O (20 ml) was diluted. The solid obtained is filtered off, with 5ml of H ₂ O-washing and purification by column chromatography on silica gel (DCM/methanol (99:1)) gave compound 49 as a white solid (38 mg, 80%); ¹ H NMR(400MHz,DMSO-d ₆ )δ8.13(s,1H),7.69(dd,J＝7.8,1.5Hz,1H),7.39(ddd,J＝8.5,7.3,1.6Hz,1H),7.35–7.24(m,7H),7.22–7.13(m,4H),7.16–7.07(m,2H),7.05–6.96(m,2H),5.27(s,2H),4.54–4.06(m,1H),2.94–2.50(m,1H),2.48(d,J＝6.7Hz,1H),1.77–1.65(m,1H),1.61–1.38(m,2H),1.27–1.23(m,1H),1.04–0.97(m,1H),0.90–0.84(m,1H).SM(IS):502.6m/z:[M+1]。

synthesis of Compounds 51 and 52.

8- (4-Benzylpiperidine-1-carbonyl) -5-methyl-5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (51)

According to the general procedure I, starting from compound 50 (120 mg,0.447mmol, WO 201519895) and 4-benzylpiperidine (104 ml,0.581 mmol). Column chromatography: DCM/MeOH (99:1); white solid (113 mg, 59%); ¹ H NMR(400MHz,DMSO-d ₆ )δ10.30(s,1H),7.64(dd,J＝7.7,2.0Hz,1H),7.50(ddd,J＝8.2,7.3,1.8Hz,1H),7.27(dd,J＝8.3,6.5Hz,2H),7.24–7.14(m,5H),7.13–7.07(m,2H),7.04(d,J＝2.0Hz,1H),4.45-4.40(m,1H),3.66-3.6(m,1H),3.28(s,3H),2.99–2.82(m,1H),2.76–2.63(m,2H),2.52(s,1H),1.82–1.71(m,1H),1.69–1.45(m,2H),1.20–1.03(m,2H)； ¹³ C NMR(101MHz,DMSO-d ₆ )δ:168.2,168.0,152.4,145.0,140.0,132.9,132.1,131.7,131.0,129.0,128.1,126.7,125.8,123.3,122.8,119.8,119.0,117.7,42.1,40.15,39.94,,37.8,37.5,31.9；SM(IS):426.1m/z:[M+1]。

8- (4-Benzylpiperidine-1-carbonyl) -5, 10-dimethyl-5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepine

11-one (52)

Compound 51 (50 mg,0.117 mmol) was dissolved in 1ml DMF and Cs was then added at room temperature ₂ CO ₃ (76 mg,0.235 mg) followed by MeI (14 ml,0.235 mmol). The reaction mixture was stirred at room temperature for 2H, then with H ₂ O (20 ml) was diluted. The solid obtained is filtered off, with 5ml of H ₂ O-washing and purification by column chromatography on silica gel (DCM/methanol (99:1)) gave compound 52 as a white solid (33 mg, 65%); ¹ H NMR(400MHz,DMSO-d ₆ )δ7.62(dd,J＝7.7,1.7Hz,1H),7.46(ddd,J＝8.7,7.7,1.9Hz,1H),7.33(d,J＝1.9Hz,1H),7.31–7.24(m,3H),7.20–7.14(m,5H),7.09(td,J＝7.5,1.0Hz,1H),4.53–4.23(m,1H),3.60–3.57(m,1H),3.43(s,3H),3.35(s,3H),2.99–2.93(m,1H),2.72–2.63(m,1H),2.52(s,2H),1.85–1.69(m,1H),1.68–1.43(m,2H),1.20–1.05(m,2H)； ¹³ CNMR(101MHz,DMSO)δ167.8,167.5,153.0,147.9,140.0,136.5,132.4,131.5,129.0,128.1,126.6,125.8,124.2,122.8,121.7,118.7,116.6,42.0,,37.4,37.4,37.0,31.9；SM(IS):m/z:440.5[M+1]。

synthesis of Compound 58.

2- ((2-amino-4- (methoxycarbonyl) phenyl) amino) -5-bromobenzoic acid methyl ester (55)

To a solution of methyl 3, 4-diaminobenzoate 54 (2.051 g,6.01 mmol) in chlorobenzene (20 mL) was added methyl 5-bromo-2-iodobenzoate 53 (1 g,6.01 mmol), K ₂ CO ₃ (0.87 g,6.30 mmol) and Cu (0.382 g,6.01 mmol). The mixture was heated to reflux for 18 hours. The mixture was filtered through a thin layer of celite while hot, and the filter cake was washed with dichloromethane. The filtrate was concentrated and the crude product purified by flash chromatography on silica gel with 10% to 100% ch ₂ Cl ₂ Gradient elution with hexane to giveTitle compound 55 (1.1 g, 50%). ¹ H NMR (400 MHz, chloroform-d) δ9.14 (bs, 1H), 8.09 (d, j=2.4 hz, 1H), 7.49 (s, 1H), 7.43 (d, j=8.2 hz, 1H), 7.38 (dd, j=9.0, 2.5hz, 1H), 7.21 (d, j=8.2 hz, 1H), 6.72 (d, j=9.0 hz, 1H), 3.93 (s, 3H), 3.90 (s, 3H) C ₁₆ H ₁₅ BrN ₂ O ₄ LR-MS calculated 378.02, found 379.3,381.3.

2-bromo-11-oxo-10, 11-dihydro-5H-dibenzo [ b, e ] [1,4] diazepine-8-carboxylic acid methyl ester (56)

To compound 55 (0.72 g,1.9 mmol) in methanol (35 mL) was added concentrated HCl (7 mL) and the mixture was heated to reflux overnight. After cooling to room temperature, the reaction mixture was filtered and the filter cake was washed with water to give the title compound 56 (0.63 g, 96%). ¹ H NMR(400MHz,DMSO-d ₆ )δ10.09(s,1H),8.52(s,1H),7.78(d,J＝2.5Hz,1H),7.59–7.52(m,3H),7.05(d,J＝8.2Hz,1H),6.96(d,J＝8.6Hz,1H),3.80(s,3H).C ₁₅ H ₁₁ BrN ₂ O ₃ LR-MS calculated 345.99, found 347.0,349.0.

2-bromo-11-oxo-10, 11-dihydro-5H-dibenzo [ b, e ] [1,4] diazepine-8-carboxylic acid (57)

To compound 4 (0.6 g,1.7 mmol) in THF: H at room temperature ₂ To a stirred solution of O (7:3, 45 mL) was added lithium hydroxide monohydrate (0.435 g,10.4 mmol). The resulting solution was stirred at 65℃for 4h. The reaction was monitored by TLC and after completion of the reaction, volatiles were removed in vacuo. The pH of the residue was acidified to about 4 with 2N HCl. The precipitated solid was filtered, washed with water (20 mL) and dried under vacuum to give compound 5 (0.570 g, 99%). ¹ H NMR(400MHz,DMSO-d ₆ )δ12.65(s,1H),10.00(s,1H),8.62(s,1H),7.70(d,J＝2.5Hz,1H),7.50–7.43(m,3H),7.04(d,J＝8.3Hz,1H),6.99(d,J＝8.7Hz,1H).C ₁₉ H ₉ BrN ₂ O ₃ LR-MS calculated 331.97, found 333.3,335.3.

8- (4-Benzylpiperidine-1-carbonyl) -2-bromo-5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (58)

To a solution of compound 57 (0.3 g,0.90 mmol) in 5ml DMF was added N- (3-dimethylaminopropyl) -N' -ethylcarboDiimine hydrochloride (EDCI) (0.276 g,1.44 mmol), N-hydroxybenzotriazole (HOBt) (0.194 g,1.44 mmol), 4-benzylpiperidine (0.205 mL,1.17 mmol) followed by DIPEA (0.470 mL,2.70 mmol). The reaction mixture was stirred at room temperature for 16 hours, quenched with water, and then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was then suspended in 3mL of ethyl acetate, after which 30mL of hexane was added. The precipitate was filtered and washed with hexane (10 mL) to give the title compound 58 (0.430 g, 97%). ¹ H NMR (400 MHz, methanol-d) ₄ )δ7.85(d,J＝2.4Hz,1H),7.44(dd,J＝8.6,2.5Hz,1H),7.28–7.23(m,2H),7.18–7.14(m,3H),7.06–6.97(m,3H),6.85(d,J＝8.6Hz,1H),4.63–4.44(m,1H),3.88–3.63(m,1H),3.11–2.92(m,1H),2.91–2.08(m,1H),2.86–2.69(m,1H),2.56(d,J＝5.7Hz,2H),1.91–1.54(m,3H),1.29–1.11(m,2H)。C ₂₆ H ₂₄ BrN ₃ O ₂ LR-MS calculated values of (c): 489.10, found: 490.0,491.9.

Synthesis of Compound 59

8- (4-Benzylpiperidine-1-carbonyl) -2-vinyl-5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (59)

Compound 58 (0.1 g,0.204 mmol) and potassium vinyltrifluoroborate (36 mg,0.265 mmol), [1,1' -bis (diphenylphosphino) ferrocene]Palladium (II) dichloride (0.030, 0.04 mmol) and K ₃ PO ₄ ·3H ₂ A mixture of O (0.143 g,0.674 mmol) in DME: H ₂ O (2:1, 4 mL) was heated to reflux for 16 hours. After cooling, the reaction mixture was partitioned between ethyl acetate and water. The ethyl acetate layer was washed with brine, dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel using CH ₂ Cl ₂ MeOH elution afforded the title compound 59 (50 mg, 50%). ¹ H NMR (400 MHz, methanol-d) ₄ )δ7.82(d,J＝2.2Hz,1H),7.47(dd,J＝8.4,2.2Hz,1H),7.29-7.24(m,2H),7.19–7.15(m,3H),7.06–6.98(m,3H),6.91(d,J＝8.4Hz,1H),6.65(dd,J＝17.6,11.0Hz,1H),5.68(dd,J＝17.6,0.9Hz,1H),5.16(dd,J＝10.9,0.9Hz,1H),4.62-4.45(m,1H),3.85-3.70(m,1H),3.12–2.94(m,1H),2.86-2.69(m,1H),2.58(d,J＝7.1Hz,2H),1.88–1.55(m,3H),1.29–1.11(m,2H).C ₂₈ H ₂₇ N ₃ O ₂ LR-MS calculated 437.21, found 438.5.

Synthesis of Compounds 60-66.

8- (4-Benzylpiperidin-1-carbonyl) -2- (piperidin-3-yl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (60)

Compound 58 (0.025 g,0.051 mmol) and 1, 3-propanediol 3-pyridineboronic acid ester (0.013, 0.076 mmol), pd ₂ (dba) ₃ CHCl ₃ (5.2 mg,0.005 mmol), X-Phos (14.6 mg,0.030 mmol) and K ₃ PO ₄ 3H ₂ A mixture of O (0.035 g,0.168 mmol) in DME: H ₂ O (2:1, 4 mL) was heated to reflux for 48 hours. After the reaction was cooled to room temperature, the mixture was partitioned between ethyl acetate and water. The ethyl acetate layer was washed with brine, dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel using CH ₂ Cl ₂ MeOH elution gave the title compound 59 (19 mg, 78%). ¹ H NMR(400MHz,DMSO-d ₆ )δ10.04(s,1H),8.83(d,J＝2.6Hz,1H),8.53(dd,J＝4.8,1.6Hz,1H),8.32(s,1H),8.03–7.99(m,2H),7.76(dd,J＝8.4,2.4Hz,1H),7.48–7.44(m,1H),7.30–7.27(m,2H),7.20–7.12(m,4H),7.05–6.99(m,3H),4.50–4.18(m,1H),3.85–3.50(m,1H),3.05–2.58(m,2H),2.53(d,J＝7.7Hz,2H),1.84–1.71(m,1H),1.70–1.41(m,2H),1.18–1.06(m,2H)。C ₃₁ H ₂₈ N ₄ O ₂ LR-MS calculated 488.22, found 489.2.

4- (8- (4-Benzylpiperidine-1-carbonyl) -11-oxo-10, 11-dihydro-5H-dibenzo [ b, e ] [1,4] diazepin-2-yl) benzamide (61)

Compound 61 was synthesized (63% yield) following the chemical reaction used to prepare compound 60 by substituting 4-aminocarbonylphenylboronic acid for 3-pyridineboronic acid 1, 3-propanediol ester. ¹ H NMR(400MHz,DMSO-d ₆ )δ10.03(s,1H),8.31(s,1H),8.09–7.91(m,4H),7.77(dd,J＝8.5,2.4Hz,1H),7.69(d,J＝8.5Hz,2H),7.38(s,1H),7.32–7.25(m,2H),7.22–7.09(m,4H),7.07–6.97(m,3H),4.58–4.14(m,1H),3.85–3.54(m,1H),3.05–2.53(m,4H),1.87–1.69(m,1H),1.68–1.43(s,2H),1.19–1.06(m,2H)。C ₃₃ H ₃₀ N ₄ O ₃ Is calculated for LR-MS 530.23, found 531.2.

4- (8- (4-Benzylpiperidine-1-carbonyl) -11-oxo-10, 11-dihydro-5H-dibenzo [ b, e ] [1,4] diazepin-2-yl) benzoic acid methyl ester (62)

Compound 62 was synthesized (23% yield) following the chemical reaction used to prepare compound 60 by substituting 4-methoxycarbonylphenyl boronic acid for 3-pyridineboronic acid 1, 3-propanediol ester. ¹ H NMR(400MHz,DMSO-d ₆ )δ10.04(s,1H),8.36(s,1H),8.15–7.95(m,3H),7.88–7.71(m,3H),7.41–7.22(m,2H),7.23–7.09(m,4H),7.07–6.97(m,3H),4.54–4.17(m,1H),3.87(s,3H),3.79–3.52(m,1H),3.08–2.54(m,4H),1.84–1.71(m,1H),1.67–1.45(m,2H),1.18–1.03(m,2H).C ₃₄ H ₃₁ N ₃ O ₄ LR-MS calculated 545.23, found 546.0.

8- (4-Benzylpiperidine-1-carbonyl) -2- (4- (methylsulfonyl) phenyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (63)

Compound 63 was synthesized (85% yield) following the chemical reaction used to prepare compound 60 by substituting 4- (methanesulfonyl) phenylboronic acid for 3-pyridineboronic acid 1, 3-propanediol ester. ¹ H NMR(400MHz,DMSO-d ₆ )δ10.05(d,J＝2.1Hz,1H),8.38(s,1H),8.07(d,J＝2.4Hz,1H),7.99–7.94(m,2H),7.92–7.86(m,2H),7.79(dd,J＝8.5,2.4Hz,1H),7.33–7.24(m,2H),7.21–7.11(m,4H),7.06–6.97(m,3H),4.55–4.16(m,1H),3.75–3.53(m,1H),3.24(s,3H),3.05–2.53(m,4H),1.83–1.68(m,1H),1.67–1.45(m,2H),1.18–1.03(m,2H)。C ₃₃ H ₃₁ N ₃ O ₄ LR-MS calculated for S565.20, realMeasured 566.1.

8- (4-Benzylpiperidine-1-carbonyl) -2- (4- (trifluoromethoxy) phenyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (64)

Compound 12 was synthesized (63% yield) following the chemical reaction used to prepare compound 60 by substituting 4- (trifluoromethoxy) phenylboronic acid for 1, 3-propanediol ester of 3-pyridineboronic acid. ¹ H NMR(400MHz,DMSO-d ₆ )δ10.04(s,1H),8.30(s,1H),7.98(d,J＝2.4Hz,1H),7.75–7.64(m,3H),7.49–7.35(m,2H),7.27(dd,J＝8.3,6.4Hz,2H),7.21–7.08(m,4H),7.08–6.93(m,3H),4.58–4.17(m,1H),3.83–3.48(m,1H),3.07–2.53(m,4H),1.83–1.68(m,1H),1.67–1.40(m,2H),1.17–1.05(m,2H)。C ₃₃ H ₂₈ F ₃ N ₃ O ₃ LR-MS calculated 571.20, found 572.2.

2- (benzofuran-2-yl) -8- (4-benzylpiperidine-1-carbonyl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (65)

Compound 65 was synthesized (52% yield) following the chemical reaction used to prepare compound 60 by substituting 2-benzofuranylboronic acid for 3-pyridineboronic acid 1, 3-propanediol ester. ¹ H NMR(400MHz,DMSO-d ₆ )δ10.07(s,1H),8.44(s,1H),8.25(d,J＝2.2Hz,1H),7.91(dd,J＝8.4,2.3Hz,1H),7.65–7.56(m,2H),7.38–7.22(m,5H),7.22–7.09(m,4H),7.07–6.94(m,3H),4.61–4.11(m,1H),3.87–3.51(m,1H),3.05–2.53(m,4H),1.84–1.69(m,1H),1.68–1.44(m,2H),1.26–1.00(m,2H)。C ₃₄ H ₂₉ N ₃ O ₃ LR-MS calculated 527.22, found 528.2.

8- (4-Benzylpiperidin-1-carbonyl) -2- (1H-pyrazol-4-yl) -5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (66)

Compound 66 was synthesized (62% yield) following the chemical reaction used to prepare compound 60 by substituting 1H-pyrazole-4-boronic acid for 3-pyridineboronic acid 1, 3-propanediol ester. ¹ H NMR(400MHz,DMSO-d ₆ )δ12.90(s,1H),9.98(s,1H),8.06(s,2H),7.87(d,J＝2.3Hz,2H),7.61(dd,J＝8.3,2.3Hz,1H),7.31–7.25(m,2H),7.21–7.15(m,3H),7.05–6.96(m,4H),4.68–4.17(m,1H),3.89–3.51(m,1H),3.03–2.53(m,4H),1.87–1.70(m,1H),1.67–1.37(m,2H),1.20–1.01(m,2H)。C ₂₉ H ₂₇ N ₅ O ₂ Is calculated 477.21 by LR-MS, found 478.2.

Synthesis of Compound 67.

8- (4-Benzylpiperidine-1-carbonyl) -2-ethynyl-5, 10-dihydro-11H-dibenzo [ b, e ] [1,4] diazepin-11-one (67)

Compound 58 (0.100 g,0.204 mmol), pd (PPh ₃ )Cl ₂ A mixture of (7.1 mg,0.01 mmol), cuI (1.9 mg,0.01 mmol), triphenylphosphine (10.7 mg,0.04 mmol), trimethylsilylacetylene (31L, 0.224 mmol) and diethylamine (0.29 mL,2.76 mmol) in dimethylformamide (1 mL) was heated at 120℃for 40min under microwave radiation. The reaction mixture was filtered and washed with dichloromethane. The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography on silica gel to give 63mg of trimethylsilyl protected intermediate. The intermediate was then treated with potassium carbonate (68 mg,4 mmol) in methanol (3 mL) and the reaction mixture was stirred at room temperature for 1h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue, which was purified by flash chromatography on silica gel to give the title compound 67 (46 mg,53%, in two steps). ¹ H NMR(400MHz,DMSO-d ₆ )δ9.99(s,1H),8.39(s,1H),7.75(d,J＝2.1Hz,1H),7.42(dd,J＝8.3,2.2Hz,1H),7.33–7.23(m,2H),7.21–7.09(m,3H),7.04–6.65(m,4H),4.50–4.17(m,1H),4.07(s,1H),3.83–3.48(m,1H),3.08–2.53(m,4H),1.87–1.68(m,1H),1.67–1.43(m,2H),1.19–1.01(m,2H)。C ₂₈ H ₂₅ N ₃ O ₂ LR-MS calculated 435.19, found 436.3.

Example 2

Cytotoxicity assays

Toxicity of the compounds was evaluated in human PBM, CEM (human lymphoblastic) and Huh-7 cells as previously described (see Schinazi r.f., somm adossi J.-P.,Saalmann V.,Cannon D.L.,Xie M.-Y.,Hart G.C.,Smith G.A.&Hahn E.F.Antimicrob.Agents Chemother.1990,34,1061-67). Cycloheximide was included as a positive cytotoxicity control, as well as untreated solvent-exposed cells as a negative control. Cytotoxic IC from concentration-response curves using the median-effect method previously described ₅₀ (see Chou t.—c.&Talalay P.Adv.Enzyme Regul.1984,22,27-55；Belen’kii M.S.&Schinazi r.f. anti res.1994,25, 1-11). The results are shown in table 1 below:

TABLE 1

Example 3

Luciferase-reported rora activity

Huh-7 cells were transfected with a luciferase reporter plasmid containing the miR-122 promoter (extending from the transcription initiation site to-900) of either the complete wild-type (WT) ROR alpha response element (RORE) or the mutated RORE (mut). One day (24 hours) after transfection, cells were treated with compound 1 at the indicated concentrations. Luciferase expression was measured 24 hours after treatment and normalized to renilla luciferase activity expressed from co-transfected pRL plasmid. pRL vectors providing constitutive expression of Renilla luciferase are used in combination with firefly luciferase vectors to co-transfect cells. The expression of renilla luciferase provides an internal control value to which the expression of the experimental firefly luciferase reporter gene can be normalized.

The results show that luciferase expression of compound 1 increased in a dose-dependent manner using WT RORE. The mutant RORE negates the activity of compound 1. As shown in fig. 1, these results demonstrate rorα activity of compound 1 as an agonist.

This assay can be used to evaluate other compounds described herein. In the case of compounds that increase luciferase expression they are ROR alpha agonists, whereas in the case of compounds that decrease luciferase expression they are ROR alpha antagonists (or partial agonists or allosteric inhibitors).

Example 4

Rorα -regulated microRNA expression.

Huh-7 cells were treated with 1. Mu.M Compound 1 or vehicle (DMSO) for 24 hours. Secreted microRNA levels in Huh-7 cell culture medium were analyzed by qRT-PCR and normalized against caenorhabditis elegans spinosa miR-39. miR-18 and miR-93 serve as controls for secreted microRNA, and are unaffected after addition of compound 1. The results are shown in fig. 2.

Example 5

Modulation of Th17 populations

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from four healthy donors. Four experiments were performed and analyzed by flow cytometry within three days of testing. The control group was not treated with the drug, the second group was treated with 10. Mu.M compound 1, the third untreated group was stimulated with PHA/IL-2, and the fourth group was stimulated with PHA/IL-2 and incubated with 10. Mu.M compound 1. These results, shown in FIG. 3A, indicate that Compound 1 is also active against CD4, even under PHA/IL-2 stimulation ⁺ The total viability of T cells had no effect. Furthermore, compound 1 had no effect on the Th17 population in the absence of PHA/IL-2 stimulation. As shown in fig. 3B, compound 1 reduced Th17 total population in PBM cells relative to vehicle control in the presence of PHA/IL-2 stimulation.

Example 6

Regulation of rorα regulated genes in C57BL/6 mice.

Healthy C57BL/6 mice were intraperitoneally injected with 7.5mg/kg of Compound 1 or saline control once. Mice were sacrificed at time points 1, 2 and 7 days post injection. miR-122 and Gpase 6mRNA levels were determined at each time point by qRT-PCR. MicroRNA levels were normalized to RNU6; standardizing plasma miR-122 into caenorhabditis elegans spinosa miR-39; and mRNA levels were normalized to HPRT.

These results are shown in figures 4A-E, indicating that miR-122 levels in plasma and liver increased up to 7 days post injection after administration of compound 1. Furthermore, rorα -regulated gene Gpase6 was significantly up-regulated up to 7 days after injection of compound 1.

Example 7

RORα modulation results in increased secretion of Mir-122 by C57BL/6 mice

C57BL/6 mice were fed a 50% High Fat Diet (HFD) for four weeks. The control group received 3 hydrodynamic 5 μg tail vein injections of antagomiR-control and 6 intraperitoneal injections of normal saline over three weeks. The second group was three injections of 5 μg of antagomiR-122 (reverse complement to inhibit miR-122 activity) into the tail vein of fluid dynamic within three weeks, and six injections of saline were performed intraperitoneally. The third group was given twice weekly injections of compound 1 (7.5 mg/kg) over 3 weeks, and once weekly injections of antagomiR-control. The last group was given twice weekly injections of Compound 1 (7.5 mg/kg) and once weekly injections of antagomiR-122.

As shown in FIGS. 5A-D, the secretion of miR-122 was enhanced when treated with Compound 1, and co-administration with antagomir-122 reduced it to baseline levels.

Detailed method

And (5) culturing the cells. HCC-derived human cell line: huh7 was cultured in DMEM supplemented with 10% Fetal Calf Serum (FCS), 1% penicillin/streptomycin.

A plasmid. As previously described, a human miR-122 promoter fragment (plasmid PmiR-122-900) was generated that spans a-900 bp region relative to the transcription initiation site (TSS) (1). The rora site in the promoter region was mutated by PCR using primers P1 and P2 as described previously (2). Table 2 describes all primers used to generate plasmids.

Luciferase assay. For the luciferase assay, cells grown on 24 well plates were co-transfected with a luciferase reporter plasmid (50 ng) and 1ng of Renilla luciferase vector (PRL, promega) using TransIT-LT1 (Mirus) transfection reagent (MIR 2300, madison, wis.). Firefly and Renilla luciferase activities were evaluated using a dual luciferase reporter assay system (Promega). Readings were taken three times on a Mithras LB 940 luminometer (Berthold Technologies).

RNA extraction and quantitative real-time PCR analysis. Samples from 200. Mu.L of plasma or medium were prepared using a miRNeasy mini-kit (Qiagen, valencia, calif., U.S.A.) with 2 minor modifications Total RNA (including small RNA) was isolated. First, 200. Mu.l of plasma or medium was lysed with 1ml of Qiazol solution. Next, 50pmol/l of synthetic single-stranded caenorhabditis elegans miRNA (cel-miR-39) was added as a spike-in control to monitor the extraction efficiency. The rest of the RNA extraction was performed according to the manufacturer's instructions. miRNA was eluted with 30. Mu.l RNase-free water. Total RNA, including miRNA, was isolated from cells or tissues using TRIzol reagent (Invitrogen, carlsbad, calif., USA). Using Quanta Biosciences qScript ^TM cDNA Synthesis kit (95047-100) cDNA was synthesized for mRNA analysis and qScript was used ^TM cDNA is synthesized by a microRNA cDNA synthesis kit (95107-100) for miRNA analysis. ABI 7900HT real-time PCR system and SYBR Green PCR Kit were used respectively: quanta Cat# 84018 and #84071 qRT-PCR was performed on miRNAs and mRNAs. Fold expression and statistical significance were calculated using the 2- ΔΔct method. All experiments were performed in triplicate.

Mice fed a high fat diet. C57BL/6 male mice were fed a 50% high fat diet for 8 days (Envigo, DIETTD 150235). All mice were kept in pathogen free facilities under a light/dark cycle of 12 h. The university of hilbert animal protection and ethics committee approved the study of mice.

Mice were injected with compound 1 and AntagomiR. 7.5mg/kg of Compound 1 in physiological saline and 3% DMSO was intraperitoneally injected into 7-8 week old C57BL/6 male mice or 9 month old Sgp FC male mice. Physiological saline was injected as a control. Mice were hydrodynamically injected with antagomiR-122 or antagomiR control (negative control) (5 μg/mouse in 1.5ml saline). Mice were sacrificed according to the legend describing the experimental results and liver, white fat and skeletal muscle tissue were frozen in liquid nitrogen or OCT embedded frozen blocks for further RNA and histological analysis. antagomiR was obtained from Sigma Aldrich, see table 3.

Table 2 primers for plasmid constructs.

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TABLE 3 synthetic small RNAs

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Subscript'm ' represents a 2' -O Me modified nucleotide; subscript's' represent phosphorothioate linkages; 'Chol' represents cholesterol linked by a hydroxyproline linkage.

Introduction to the invention

microRNA-122 (miR-122) is associated with the expression of FGF 21. Expression of miR-122 is dependent on inflammatory signaling, which also suggests that its secretion from the liver has a distal effect on other organs. In addition, miR-122 is also regulated by Free Fatty Acids (FFA) mediated by the ROR alpha of activated hepatocytes. miR-122 in hepatocytes is increased by FFA-ROR alpha mechanism, resulting in upregulation of FGF 21.

A set of rora agonists was developed and one compound was selected based on its activation of the miR-122 promoter. This activation results in a beneficial increase in endogenous FGF21 levels, which can be used to treat pancreatitis.

Results

RORα activation

Rorα regulates miR-122 expression in mice, which is mediated by FFA (Chai, gastroenterology, volume 153,Issue 5,November 2017,Pages 1404-1415). The level of RFD decreases upon HFD and increases upon activation with rorα activator compound 1.

In a human with decreased miR-122, the expression of the miR-122 target gene is increased. The miR-122 target gene is also inversely related to ROR alpha expression. FGF21 expression is positively correlated with pre-miR-122.

FGF21 is a known target for RORα (Wang, J Biol chem.2010May21;285 (21): 15668-73). FGF21 up-regulation in liver following cold exposure has recently been shown (Ameka, sci Rep.2019Jan 24;9 (1): 630). While not wishing to be bound by a particular theory, it is believed that this may be due to an increase in ROR alpha in cold. To evaluate this hypothesis, HUH7 human HCC cells were transfected with a rora reporter system, in which luciferase was expressed from miR-122 promoter with rora binding site. Increased levels of primiR-122 and miR-122 are associated with decreased miR-122 target genes (Aldo A and Dgat 1). Expression of miR-122 is cold-sensitive, based on the binding of ROR alpha to its consensus sequence in the miR-122 promoter. To determine whether the RORα -miR-122 mechanism is associated with humans, a human study was also conducted (Hadassah University Hospital IRB approval # HMO-0025-18). In this study, humans used the cardiopulmonary machine and systemic cooling to perform large vessel cardiovascular surgery. The expression of miR-122 was measured and a significant increase in plasma miR-122 was found following a decrease in temperature.

These results indicate that an increase in rorα activity and an increase in miR-122 promoter activity can increase FGF21 levels, which in turn has a beneficial effect on pancreatitis. For this, a panel of rora agonists was generated.

Rorα consists of an N-terminal activation function 1 (AF-1) interacting with a coactivator and a subsequent DNA-binding domain comprising two zinc finger motifs, a flexible hinge region, and a C-terminal Ligand Binding Domain (LBD) comprising hormone-responsive activation function 2 (AF-2). Binding of the agonist to RORα -LBD induces a conformational change that enables the coactivator to bind AF-2. The most potent agonist that was solubilized in coordination with rora-LBD was cholesterol sulfate (PDBID 1S 0X). The ligand binding pocket targeted to the crystal structure is screened virtually by high throughput to identify new rora agonists. Binding capacity of a proprietary library of 300,000 compounds of similar drugs was evaluated using the Schrodinger Maestro Glide HTVS workflow. Using Prime MMGBSA (allowIs the flexibility of (c) the first 200 compounds were further scored. The first 100 compounds were visually inspected and selected using a luciferase assay at the miR-122 promoter regionTwelve were selected for evaluation.

Liver and systemic effects of rorα are mediated through miR-122

To determine whether rora metabolism and biochemistry is mediated by miR-122, the following experiments were performed. Activation of compound 1 is achieved by mutating this site in the miR-122 promoter via binding/activation of rora DNA to the miR-122 promoter. After 16h exposure of HuH7 cells to compound 1, the cell level of miR-122 was unchanged, but significant miR-122 was secreted in the medium (no apparent toxicity to cells as determined by LDH release, data not shown). However, compound 1 was administered to mice for several weeks, and miR-122 levels were elevated in both liver and plasma. This is associated with increased liver precursor levels of both Pre-miR-122 and Pri-miR-122, as well as decreased AldoA (a known target for miR-122) and increased G6Pase (a known ROR alpha target gene) (Chauvet, PLoS one.2011;6 (7): e 22545). miR-122 reaches the distal tissue. To evaluate the effect of compound 1 on this distal effect of miR-122, miR-122 was measured in myocardial tissue, which showed elevated levels of mature miR-122, while the three miR-122 target genes were down-regulated with each other. Following administration of compound 1, mature miR-122 was also identified as WAT and muscle in other organs (no levels of pri-miR-122 were detected in muscle tissue, indicating that mature miR-122 was not expressed in muscle from miR-122 promoter).

To determine whether the mechanism of action of miR-122 and compound 1 are identical on the same pathway, an experiment was designed in which both molecules were administered together as well as separately. In this study, mice were fed 50% HFD and treatment was started 4 weeks after the animals' diet to establish NASH prior to treatment. Treatment was started after 4 weeks (due to half-life extension, antagomiR was given once a week, and due to plasma t) _1/2 RS was given twice a week for 2.7 hours) and for 3 weeks. Mice were weighed starting from week 3 and treatment started after one week. Control mice (antagomiR-control once weekly, DMSO diluted in physiological saline twice weekly) steadily increased in body weight. The mice administered with antagomir-122 had the greatest weight gain. Animals treated with rora agonist compound 1, body weight stableWeight loss and weight loss. Mice administered with antagomir-122 and compound 1 had their body weight fully restored to that of the control animals. This phenomenon suggests that miR-122 and Compound 1 may antagonize each other.

A decrease in miR-122 in the liver and plasma was observed when antagomiR-122 was administered and an increase in miR-122 in both the liver and plasma was observed when compound 1 was administered. The increase in miR-122 also has an effect on its target genes (including Agpat1, dgat1 and FGF 21). Liver antagomiR-122 and compound 1 also produced a similar pattern of action on muscle as liver, possibly through miR-122 secretion.

The level of FGF21 information in the liver correlated with the level of pri-miR-122, indicating that co-regulation was present. These observations reinforce our hypothesis that controlling miR-122 levels by either reducing the level of miR-122 in the liver by antagomiR-122 or increasing the level of miR-122 in the liver by compound 1 has hepatic/central and distal/peripheral effects. The overall effect of compound 1 ultimately results in an increase in FGF21 levels, which is beneficial for treating, preventing, reducing the susceptibility to, lessening the severity of, and/or delaying the progression of pancreatitis.

Action of activating ROR alpha-miR-122-triglyceride circulation

After demonstrating that compound 1 is a clinically relevant miR-122 activator with beneficial biochemical effects, the following study was aimed at determining its effect on miR-122 precursors. Compound 1 was administered to mice with defined NASH. Activators increase miR-122 levels in mouse liver and plasma. Administration of the ROR alpha activator/agonist compound 1 compound results in an increase in miR-122 precursors and ROR alpha targets. These results indicate that the activator does function in the model.

Anti-inflammatory and antifibrotic effects of activating rora-miR-122-triglyceride circulation with compound 1

Once the favorable metabolic profile of rora activator compound 1 is observed, the effect of compound 1 on liver inflammation and fibrosis can be determined. The effect of compound 1 on liver inflammation and fibrosis was evaluated in a mouse atherogenic diet model (Anavi, lab invest.2015Aug;95 (8): 914-24). After the development of liver inflammation and fibrosis at week 3 of diet, animals began to receive compound 1. After an additional 3.5 weeks, where animals received 3 compound 1 per week, many endpoints of the animals were assessed. Compound 1 significantly improved liver enzymes. It was demonstrated that mature miR-122 increased in both tissues and plasma after administration of Compound 1. Compound 1 significantly ameliorates liver inflammation. This improvement in inflammation is associated with a significant reduction in liver fibrosis, which can be assessed by two methods, masson trichromatic staining and αsma staining. As shown by CD34 staining of the livers of these mice (data not shown), compound 1 had no effect on liver blood vessels. The effect of rora activator compound 1 is also evident for fibrosis driving genes. FGF21 is also associated with inflammation and fibrosis in the pancreas, so the effect of compound 1 on FGF21 levels, and thus on liver inflammation and fibrosis, can be inferred to be treatment of pancreatitis and its prevention, to reduce the susceptibility of pancreatitis, to reduce the severity of pancreatitis, or to delay the progression of pancreatitis.

Discussion of the invention

Activating rorα has a major beneficial effect on pancreatitis. The beneficial effects of rora on pancreatitis are mediated through mature miR-122, although other rora activities may contribute to these beneficial effects. The role of miR-122 is by targeting the expression of a central enzyme in TG biosynthesis. According to the evidence, it is shown that ROR alpha activates miR-122 expression and increases its secretion into plasma, reaching WAT, muscle and myocardium, accelerating its distal effects, which we explain as the effect of ROR alpha activation is found in both liver and whole body.

In order to control the activation of ROR alpha and enhance its potentially beneficial effects, a screening system was developed to identify compounds that enhance the activity of ROR alpha on miR-122 expression. We have identified a compound (compound 1) that has potential therapeutic effects in enhancing FGF21 levels. Interestingly, compound 1, which increases miR-122 expression and secretion, also showed significant metabolic effects, further demonstrating its effectiveness in treating the subpopulation of pancreatitis caused by metabolic disorders.

Many previous reports, as well as our reports, all indicate the possibility of "hijacking" miR-122 in the liver as a mechanism action for anti-lipotoxic effectors. The mechanism of miR-122 production in the liver is powerful. Each hepatocyte stores 250,000 copies of miR-122 and miR-122 (Simerzin, hepatology.2016Nov;64 (5): 1623-1636). The effective distal activity of miR-122 depends on the high yield and secretion of miR-122 to produce high plasma levels. Such high productivity suggests that miR-122 can translate into an effective therapeutic compound. However, in connection with its development of a system for synthesizing and preparing a drug from synthetic miR-122 (mimic-miR-122) and injecting it into NASH patients for many years, it would be preferable to develop a small drug that can induce expression of liver endogenous miR-122 and can be administered daily to patients. miR-122 is also expressed and secreted by TNF alpha signaling. However, tnfα injection is not relevant to the clinical setting of NASH. miR-122 also has other therapeutic properties, including increased expression of FGF 21.

The data shown in this report indicate that rora activation has significant activity, which increases miR-122 expression and reaches other organs, both in the liver and in other organs (including the pancreas). Thus, rora activators are proposed as promising compounds to be developed and evaluated for their clinically beneficial effects on pancreatitis in patients.

Materials and methods

Cell culture

Human hepatoma cell line-Huh 7 was cultured in DMEM supplemented with 10% Foetal Calf Serum (FCS), 1% penicillin/streptomycin (Thermo Scientific, waltham, mass., USA). The cells were incubated at 37℃in the presence of 5% CO ₂ Except for the experiments in which the cells were placed at 32℃as indicated herein.

Real-time RT-PCR for RNA extraction and quantification

Total RNA (including small RNA) was isolated from 200. Mu.L of plasma or medium samples using a miRNeasy mini-kit (Qiagen, valencia, calif., U.S.A.) with 2 minor modifications. First, 200. Mu.l of plasma or medium was lysed with 1ml of Qiazol solution. Next, 50pmol/l of synthetic single-stranded caenorhabditis elegans miRNA (cel-miR-39) was added asA labeling (spike-in) control was added to monitor extraction efficiency. The rest of the RNA extraction was performed according to the manufacturer's instructions. miRNA was eluted with 30. Mu.l RNase-free water. Total RNA, including miRNA, was isolated from cells or tissues using TRIzol reagent (Invitrogen, carlsbad, calif., USA). Using Quanta Biosciences qScript ^TM cDNA Synthesis kit (95047-100) cDNA was synthesized for mRNA analysis and qScript was used ^TM cDNA is synthesized by a microRNA cDNA synthesis kit (95107-100) for miRNA analysis. ABI 7900HT real-time PCR system and SYBR Green PCR Kit were used respectively: quanta Cat# 84018 and #84071 qRT-PCR was performed on miRNAs and mRNAs. Fold expression and statistical significance were calculated using the 2- ΔΔct method. All experiments were performed in triplicate. The primers used for qRT-PCR are shown in Table 4.

Plasmid(s)

As previously described, human miR-122 promoter fragments (plasmids PmiR-122-900 and PmiR-122-RORαmut, respectively) spanning the-900 bp region relative to the Transcription Start Site (TSS) and mutating the RORα binding site were generated.

Transfection

For the luciferase assay, cells grown on 24 well plates were co-transfected with a luciferase reporter plasmid (50 ng) and 1ng of Renilla luciferase vector (PRL, promega) using liposome LTX (Invitrogen) transfection reagent. For all experiments, transfection was performed using serum-free medium (Opti-MEM; cat #31985070;Thermo Scientific).

Luciferase Activity assay

After transfection, cells were lysed with passive lysis buffer (Cat#E 1941; promega), shaken for 20min at room temperature, and transferred to a suitable 96-well plate. Firefly and Renilla luciferase activities were evaluated on a photometer Mithres 2000 (Centro XZ, LB960, berthold Technologies, bad Wildbad, germany) using a dual luciferase reporter assay system (Cat#E1910; promega). Normalizing the luciferase activity to a renilla luciferase activity. Three readings were taken.

Rora agonist treatment

Commercial rora agonist SR1078 (Cayman Chemical) and rora compound stock solutions were prepared by dissolving in DMSO (1 mg/ml). Huh7 cells were treated overnight with 5 μm SR1078 or 1 μm of all other tested compounds. DMSO alone (0.2%) was used as control. Rora agonists, compound 1, are dissolved in physiological saline and up to 5% dmso and injected intraperitoneally into mice according to the text dose. The triglycerides, free fatty acids and beta-hydroxybutyrate were quantified.

To determine liver and lipid content of the liver, the muscle and liver tissue (40-80 mg) were homogenized in 0.5ml of chloroform: tris solution (v/v, 1:1), the homogenate was transferred to 1ml of chloroform: methanol solution (v/v, 2:1) and centrifuged at 3000g (at-2 ℃) for 10min (Heraeus Megafuge 16R centrifuge). The organic phase was mixed with 5% triton x100 in chloroform, dried and redissolved in water. After lipid extraction, triglyceride (TG) concentration in the samples was measured using a triglyceride quantification kit (BioVision) according to the manufacturer's instructions. Plasma free fatty acids and β -hydroxybutyrate were measured directly from plasma samples using a commercial colorimetric kit (BioVision).

Animal study

Male C57BL/6 mice 7-8 weeks old were purchased from Harlan Laboratories (Yes-cold, israel). All mice were kept in pathogen free facilities under a light/dark cycle of 12 h. Mice were treated according to the criteria outlined in "guidelines for care and use of laboratory animals" written by the national academy of sciences and published by the national institutes of health. The university of hilbert animal protection and ethics committee approved the study of mice; ethical number MD-15-14423-3.

antagomiR-122 treatment of High Fat Diet (HFD) fed mice

C57BL/6 mice, 7-8 weeks old, were fed a diet consisting of 50% fat, 20% sucrose, 10% fructose, 1.25% cholesterol (Envigo, td.150235) or 50% hfd for 4 weeks. In experiments with miR-122 inhibition by antagomiR, mice were hydrodynamically injected with antagomiR-122 or antagomiR-control (5 μg/mouse in 1.5mL saline) for 4 weeks once weekly, and still fed HFD or feed. After 4 weeks of injection, mice were sacrificed and liver, white fat and skeletal muscle tissue were frozen in liquid nitrogen or in frozen blocks embedded at optimal cutting temperatures for further RNA and histological analysis. antagomiR was purchased from Sigma-Aldrich (St Louis, MO); see table 5.

Compound 1 and anti-agomiR-122 treatment of mice fed HFD or atherogenic diet

Male C57BL/6J mice 7-8 weeks old were randomly housed in standard cages and fed HFD or atherogenic diet (consisting of 1% cholesterol and 0.5% cholic acid, see also Table 6). During the experiment, all mice were free to drink water. Body weight was monitored every 3 days during feeding. In HFD experiments, after 4 or 6 weeks, the resulting obese mice were treated with antagomiR-122 (5 μg/mouse, once per week for 3 weeks), or injected intraperitoneally with compound 1 (RORα agonist, twice per week, 7.5mg/kg for 3 weeks, or 3 times per week, 15mg/kg for 3 weeks). The obese control (HFD) group was administered with saline, DMSO, and antagomiR controls only. After 3 weeks of treatment, mice were sacrificed and livers were removed for RNA-seq analysis. Liver, white fat and skeletal muscle tissue were frozen in liquid nitrogen or in frozen blocks embedded at optimal cutting temperatures for further RNA and histological analysis. In the atherogenic diet experiment, mice were treated with 15mg/kg of compound 1 after 3 weeks of diet. After 3.5 weeks of treatment, mice were sacrificed and livers were frozen in liquid nitrogen or in frozen blocks embedded at optimal cutting temperature. Plasma was collected from atherogenic diet-fed mice and stored at-20℃for use The analyzer and test strip (Roche) performed ALT and AST analyses.

Multi-parameter metabolic assessment

The metabolic and motor status of mice was measured by using a Prometion high definition behavioral phenotype system (Sable Instruments, inc., las Vegas, NV, USA), which is a systemA multiparameter assessment comprising subsystems for open circuit indirect calorimetry, feeding, water intake, activity, running wheel and weight measurement in a conventional home cage minimizing stress. Data acquisition and instrument control was performed using MetaScreen software version 2.2.18.0 and raw data obtained was processed using ExpeData 1.8.4 using analysis scripts detailing aspects of data conversion. C57BL/6 mice were fed with HFD for 6 weeks, then treated with 15mg/kg of compound 1 3 times per week for 2 weeks, then placed in the metabolic chamber, fed ad libitum and drinking water, and received a standard 12h dark/12 h dark period, including a 24h acclimation period and then a sampling duration of 48 h. The breathing GAs was measured by using a GA-3 GAs analyzer (noble Systems inc., las Vegas, NV, usa) using a pull mode negative pressure system the GAs flow was measured and controlled by FR-8 (noble Systems inc., las Vegas, NV, usa), the flow rate was set to 2000mL/min the water vapor was continuously measured and its pair O was compensated mathematically ₂ And CO ₂ Is used for dilution. Effective mass was calculated by ANCOVA analysis. Respiratory Quotient (RQ) is calculated as VCO ₂ /VO ₂ Ratio of the two components. Total energy consumption (TEE) calculated as VO ₂ x (3.815+1.232 xRQ), normalized to effective body weight, and expressed as kcal/h/kgoff. Fat Oxidation (FO) and carbohydrate oxidation (CHO) are calculated as: fo=1.69 x VO ₂ –1.69x VCO ₂ Cho=4.57 x VCO ₂ –3.23x VO ₂ And expressed as g/d/kgiff. Dynamic activity and position were monitored using an XYZ beam array and at a beam pitch of 0.25cm, while calorimetric data was collected.

Oil red O dyeing

Liver tissue was embedded in the optimal cutting temperature gel and cut into 10 μm frozen sections. For oil red O staining, stock solutions of oil red O (Sigma-Aldrich) (1 g/10mL in propylene glycol), filtration and protection from light were prepared. The frozen sections were immersed in formalin, stained with oil red O for 15min, and counterstained with hematoxylin for 30 seconds.

Human blood sample and heparin elimination

For measuring miR-122, pairFFA and human FGF21 (abcam) analyses were performed using a cardiopulmonary machine and a blood sample taken from a patient undergoing a large vessel cardiovascular procedure with systemic cooling. This was done under the approval of the Hadassah hospital IRB Committee approval number 0025-18-HMO. All subjects obtained informed consent and permission for the study using the biological material. Tube No. 2 represents the time during surgery before the patient cools down, and tube No. 3 represents the time during surgery at which the body temperature is at its lowest. According to the previously described scheme ^3,4 Heparin was removed from the RNA solution isolated from patient plasma samples, and briefly, 5. Mu.L of the RNA sample was combined in water with 5. Mu.L of heparinase working solution (0.085 IU/mL heparin I (Sigma-Aldrich; catalog number H2519), 2000 units/mL riboLock RNase inhibitor (Life Technologies; catalog number EO 0381), 10mmol/L Tris HCl pH 7.5, 2mmol/L CaCl) ₂ 25mmol/L NaCl) and kept at 25℃for 3 hours. After the reaction, the sample was used directly as an RNA template for reverse transcription.

Histology and immunohistochemistry of tissues

Liver and fat samples were placed in 4% buffered formaldehyde for 24 hours, then in 80% ethanol, then embedded in paraffin blocks. Liver and adipose tissue were cut into 5mm sections, deparaffinized with xylene and hydrated by fractionated ethanol. For H & E staining, tissue sections were stained with hematoxylin (Emmonya Biotech ltd.) and eosin (Leica, surgiath). Liver macrophages were stained with rat anti-mouse F4/80 antigen (Serotec), then anti-rat HRP (Histofine), and visualized with DAB kit (Zymed). Liver sections were stained with Masson trichrome (Sigma). Liver CD3+ T cells were stained with rat anti-human CD3 antibody (Bio-Rad) followed by anti-rat HRP (Histofine) and visualized with AEC (Invitrogen). alpha-SMA positive cells were stained with mouse anti-human smooth muscle actin antibody (Dako), then stained with anti-mouse HRP (Dako) and visualized with DAB. The percentage of positive staining area per high power field was calculated in 5-10 random fields by ImageJ software.

Statistical analysis

Statistical analysis WAs performed on the data using Excel software packages (Microsoft, redmond, WA) or GraphPad Prism6 (GraphPad Software inc., la Jolla, CA). The difference between the two groups was determined using a two-tailed Student t-test and Pearson and Spearman correlation coefficients. Data are given as mean ± SD and shown as error bars for all experiments. The difference was considered significant at P < 0.05. Reported data were obtained from at least 3 biological replicates.

TABLE 4 primers for real-time PCR

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Table 5. The antagomiR sequences used in the study. All oligonucleotides were synthesized by IDT (IDT, coralville, IA, usa). Chemical modification of antisense oligonucleotides: subscript'm ' represents a 2' -OMe modified nucleotide; subscript's' represent phosphorothioate linkages; 'Chol' represents cholesterol linked by a hydroxyproline linkage.

Table 6. Normal diet and atherogenic diet compositions.

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Reference is made to:

1.Chai,C.et al.Metabolic Circuit Involving Free Fatty Acids,microRNA 122,and Triglyceride Synthesis in Liver and Muscle Tissues.Gastroenterology 153,1404–1415(2017).

2.Rivkin,M.et al.Inflammation-Induced Expression and Secretion of MicroRNA 122Leads to Reduced Blood Levels of Kidney-derived Erythropoietin and Anemia.Gastroenterology(2016).doi:10.1053/j.gastro.2016.07.031

3.Kondratov,K.et al.Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs.Biomol.Detect.Quantif.8,9–14(2016).

4.Izraeli,S.,Pfleiderer,C.&Lion,T.Detection of gene expression by PCR amplification of RNA derived from frozen heparinized whole blood.Nucleic Acids Res.19,6051(1991).

example 8

Demonstration of Compound 1 as a potent ROR alpha agonist and increases FGF21 expression

Experiment design: c57BL/6 mice were fed a High Fat Diet (HFD) for 6 weeks, 3 times a week with 15mg/kg of compound 1 (or physiological saline + DMSO) for 3 weeks (n=6).

FIGS. 7A and 7B show the results of qRT-PCR analysis of miR-122 extracted from plasma and liver in mice treated with Compound 1 or physiological saline, respectively. FIG. 7C shows qRT-PCR analysis of ROR alpha target genes pri-miR-122 and pre-miR-122mRNA extracted from mouse liver.

Treatment with compound 1 induced expression and secretion of miR-122 and precursors in plasma and liver. In addition, treatment with compound 1 significantly induced expression of rorα -regulated genes FGF21 and Gpase 6.

Example 9

Rora agonist compound 1 improves markers of liver injury and fibrosis in a fibrotic diet mouse model.

Experiment design: c57BL/6 mice were fed an atherogenic diet (inducing fibrosis) for 3 weeks, and 15mg/kg of compound 1 (or physiological saline+dmso) was injected 3 times per week for 3.5 weeks (n=8).

The results are shown in FIGS. 8A-D. qRT-PCR analysis of miR-122 extracted from 8A) plasma and 8B) liver for untreated (grey bars) and treated (black bars) groups. miR-93 and miR-18 were used as negative controls in plasma and liver, respectively. 8C) ALT and AST plasma levels measured at the end of the experiment. 8D) qRT-PCR analysis of mRNA of gene involved in fibrosis and ROR alpha target gene (FGF 21) extracted from mouse liver. Normalizing the microRNA level in the blood plasma to caenorhabditis elegans spinosa miR-39; microRNA levels in tissues were normalized to RNU6.mRNA levels were normalized to HPRT. Data are represented by error line = SD. * P <.05, P <.01.* P < 001, P <0.0001.

The effect of compound 1 on liver inflammation and fibrosis was measured. The effect of compound 1 on liver inflammation and fibrosis in a mouse atherogenic diet model has been evaluated. After the development of liver inflammation and fibrosis at week 3 of diet, animals began to receive compound 1. After an additional 3.5 weeks, where animals received 3 compound 1 per week, many endpoints of the animals were assessed. We confirmed that mature miR-122 increased in both tissues and plasma following administration of compound 1. Treatment with compound 1 significantly improved biomarkers of liver injury (AST and ALT) in addition to biomarkers that reduced inflammation (Tgfb 2 and TgfbR 2) and fibrosis (Acta 1, col1A1 and Col3 A1).

Example 10

Rora agonist compound 1 ameliorates liver inflammatory features in a fibrotic diet mouse model.

Experiment design: c57BL/6 mice were fed an atherogenic diet (inducing fibrosis) for 3 weeks, and 15mg/kg of compound 1 (or physiological saline+dmso) was injected 3 times per week for 3.5 weeks (n=8). FIG. 9A shows representative photomicrographs of H & E, CD3 and F4/80 stained livers taken from normal saline or compound 1 treated mice, with scale bars representing 10 μm. The chart shown in FIG. 9B demonstrates quantification of positively stained F4/80 regions using ImageJ.

Mice treated with compound 1 showed reduced immune infiltration by H & E staining, reduced T cell density by CD3 staining, and reduced level of myeloid infiltration by F4/80 staining. These results demonstrate that compound 1 exhibits anti-inflammatory effects.

Example 11

Rora agonist compound 1 reduced liver fibrosis in a dietary mouse model of fibrosis.

Experiment design: c57BL/6 mice were fed an atherogenic diet (inducing fibrosis) for 3 weeks, and 15mg/kg of compound 1 (or physiological saline+dmso) was injected 3 times per week for 3.5 weeks (n=8). The results are shown in FIGS. 10A-D.

Fig. 10A and C are representative photomicrographs of Masson trichromatic (m.t.) stained and alpha-SMA stained livers taken from saline or compound 1 treated mice, with scale bars representing 10 μm. Fig. 10B and 10D are graphs showing quantification of positively stained areas using ImageJ.

Experiment design: c57BL/6 mice were fed an atherogenic diet (inducing fibrosis) for 3 weeks, and 15mg/kg of compound 1 (or physiological saline+dmso) was injected 3 times per week for 3.5 weeks (n=8). The results are shown in FIGS. 10A-D.

The effect of compound 1 on liver fibrosis (Masson trichrome and a-SMA) was evaluated using two stains. Using both staining methods, the untreated group showed a larger positive area, whereas treatment with compound 1 significantly reduced the fibrotic area by a factor of 5 (M.T) and 7 (α -SMA). These observations strongly confirm that compound 1 exhibits anti-fibrotic activity in this mouse model.

Example 12

Rora agonists increase promoter microRNA 122 (MIR 122) activity and fibroblast growth factor 21 (FGF 21) expression.

The following examples were conducted to show that identified RORA (ROR-a) agonists increase expression of MIR122 promoter activity and FGF21 expression. FGF21 itself is useful for the treatment of pancreatitis (Hernandez et al, sci. Transl. Med.12, eaay5186 (2020)). However, FGF21 is administered by injection, and thus there is a need to identify small molecules that can be administered orally and to increase endogenous levels of FGF21, rather than relying on injection of FGF21, particularly for long term administration.

As discussed in this example, compounds that are ROR alpha agonists have been identified and these compounds increase the expression of MIR122 and FGF 21. Thus, the compounds increase endogenous production of FGF21 and are useful for treating pancreatitis.

The method involves screening a chemical library to identify ROR alpha agonists. The effect of these compounds was evaluated on the human hepatocellular carcinoma cell line (Huh 7). C57BL/6 mice were fed food or high fat diet for 4 weeks to induce fatty liver. Mice were given weekly fluid dynamic tail vein injections of MIR122 antagonist (antagomiR-122) or control antagomiR for 3 weeks, or intraperitoneal injections of rora agonist compound 1 or vehicle twice weekly for 3 weeks while still on HFD or food diet. Liver, gonadal white fat and skeletal muscle were collected and analyzed by RT-PCR, histology and immunohistochemistry.

Another group of mice was fed an atherogenic diet, with or without compound 1 injected, for 3 weeks. Compound 1 has the general formula:

liver was analyzed by immunohistochemistry and plasma was analyzed for aminotransferase levels. We analyzed liver tissue data for NASH patients in RNAseq databases GSE33814 and GSE 89632.

As a result, mice injected with antagomiR-122 significantly reduced MIR122 levels in plasma, liver and white adipose tissue.

Compound 1 was identified as a rora agonist and found to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed HFD or atherogenic diet, injection of compound 1 increased liver levels of MIR122 precursors and decreased liver synthesis of triglycerides by decreasing expression of biosynthetic enzymes.

Plasma MIR122 levels were increased in patients undergoing cardiovascular surgery compared to pre-surgery; an increase in plasma MIR122 expression correlates with an increase in rorα levels.

Conclusion compound 1 is an agonist of rora, which increases MIR122 expression in cell lines and mouse liver. Rora agonists may be developed for the treatment of pancreatitis and other conditions mediated by FGF 21.

Introduction to the invention

Liver-specific microRNA-122 (MIR 122) is associated with liver lipid metabolism. MIR122 is induced by Free Fatty Acids (FFAs), which induction is mediated by activation of liver rorα. Increasing liver MIR122 via FFA-rora mechanism results in inhibition of Triglycerides (TG) due to targeting and lowering of MIR 1228's enzyme levels involved in triglyceride biosynthesis.

These and other effects caused by the increase in MIR122 result in a determination of whether there are other beneficial effects of activating rorα. To this end, we designed a new set of rora agonists and selected a compound based on its remarkable effect of activating the MIR122 promoter. This activation has a beneficial effect on the liver of the mouse model, including reversal of fibrosis.

Materials and methods

Cell culture

Human hepatocellular carcinoma cell line Huh7 was cultured in DMEM supplemented with 10% Fetal Calf Serum (FCS), 1% penicillin/streptomycin (Thermo Scientific, waltham, ma, usa). The cells were incubated at 37℃in the presence of 5% CO ₂ Except for the experiments in which the cells were placed at 32℃as indicated herein.

Plasmid(s)

The human MIR122 promoter fragment spans the region relative to the transcription initiation site (TSS) -900bp, and the ROR alpha binding site was mutated (plasmids pMIR122-900 and pMIR122-ROR alpha mut, respectively) as described previously (Chai C. Et al Metabolic Circuit Involving Free Fatty Acids, microRNA 122,and Triglyceride Synthesis in Liver and Muscle Tissues.Gastroenterology 153,1404-1415 (2017) and Rivkin M. Et al information-Induced Expression and Secretion of MicroRNA 122-Leads to Reduced Blood Levels of Kidney-Derived Erythropoietin and Anemia. Gastroenterology 151,999-1010e3 (2016)).

Transfection

For the luciferase assay, cells grown on 24 well plates were co-transfected with a luciferase reporter plasmid (50 ng) and 1ng of Renilla luciferase vector (PRL, promega) using liposome LTX (Invitrogen) transfection reagent. For all experiments, transfection was performed using serum-free medium (Opti-MEM; cat #31985070;Thermo Scientific).

Luciferase Activity assay

After transfection, cells were lysed with passive lysis buffer (Cat#E 1941; promega), shaken for 20min at Room Temperature (RT) and then transferred to a suitable 96-well plate. Firefly and Renilla luciferase activities were evaluated on a photometer Mithres 2000 (Centro XZ, LB960, berthold Technologies, bad Wildbad, germany) using a dual luciferase reporter assay system (Cat#E1910; promega). Normalizing the luciferase activity to a renilla luciferase activity. Readings were taken in triplicate.

Rora agonist treatment

Commercial rora agonist SR1078 (Cayman Chemical) and our freshly synthesized rora compound stock solution were prepared by dissolving in DMSO (1 mg/ml). Huh7 cells were treated overnight with 10 μm SR1078 or 1 μm of all other tested compounds. DMSO alone (0.2%) was used as control. Rora agonist compound 1 was dissolved in physiological saline and up to 5% dmso and injected intraperitoneally into mice according to the text dose.

RNA extraction and quantitative real-time RT-PCR (qRT-PCR)

Total RNA (including small RNA) was isolated from 200. Mu.L of plasma or medium samples using a miRNeasy mini-kit (Qiagen, valencia, calif., U.S.A.) with 2 minor modifications. First, 200. Mu.l of plasma or medium was lysed with 1ml of Qiazol solution. Next, 50pmol/L of synthetic single-stranded caenorhabditis elegans miRNA (C.elegans miR-39) was added as a spike-in control to monitor the extraction efficiency. The rest of the RNA extraction was performed according to the manufacturer's instructions. miRNA was eluted with 30. Mu.l RNase-free water.

Total RNA, including miRNA, was isolated from cells or tissues using TRIzol reagent (Invitrogen, carlsbad, calif., USA). Using Quanta Biosciences qScript ^TM cDNA Synthesis kit (95047-100) cDNA was synthesized for mRNA analysis and qScript was used ^TM cDNA is synthesized by a microRNA cDNA synthesis kit (95107-100) for miRNA analysis. ABI 7900HT real-time PCR system and SYBR Green PCR Kit were used respectively: quanta Cat# 84018 and #84071 qRT-PCR was performed on miRNAs and mRNAs. Fold expression and statistical significance were calculated using the 2- ΔΔct method. All samples from one experiment were run in triplicate.

Animal study

Male C57BL/6 mice 7-8 weeks old were purchased from Harlan Laboratories (Yes-cold, israel). All mice were kept in pathogen free facilities under a light/dark cycle of 12 h. Mice were treated according to the criteria outlined in "guidelines for care and use of laboratory animals" written by the national academy of sciences and published by the national institutes of health. The university of hilbert animal protection and ethics committee approved the study of mice; ethical number MD-15-14423-3.

antagomiR-122 treatment of High Fat Diet (HFD) fed mice

C57BL/6 mice 7-8 weeks old were fed feed consisting of 50% fat, 20% sucrose, 10% fructose, 1.25% cholesterol (Envigo, td.150235) or 50% hfd for 4 weeks. In experiments with miR-122 inhibition by antagomiR, mice were hydrodynamically tail intravenously injected with antagomiR-122 or antagomiR-control (5 μg/mouse in 1.5mL saline) once a week for 3 weeks and still fed HFD or feed. After 3 weeks of injection, mice were sacrificed and liver, gonadal white fat and skeletal muscle tissue were frozen in liquid nitrogen or in frozen blocks embedded at optimal cutting temperatures for further RNA and histological analysis. AntagomiR was purchased from Sigma-Aldrich (St Louis, MO).

Compound 1 and antagoMIR122 treatment of mice fed HFD or atherogenic diet

Male C57BL/6J mice 7-8 weeks old were randomly housed in standard cages and fed HFD or atherogenic diet (consisting of 1% cholesterol and 0.5% cholic acid). During the experiment, all mice were free to drink water. Body weight was monitored every 3 days during feeding.

In HFD experiments, after 4 or 6 weeks, the resulting obese mice were treated with antagoMIR122 (5 μg/mouse, once per week for 3 weeks), or injected intraperitoneally with Compound 1 (ROR alpha agonist, twice per week, 7.5mg/kg for 3 weeks, or 3 times per week, 15mg/kg for 3 weeks). The obese control (HFD) group was administered with saline, DMSO, and antagomiR controls only.

After 3 weeks of treatment, mice were sacrificed and livers were removed for RNA-seq analysis. Liver, gonadal white fat and skeletal muscle tissue were frozen in liquid nitrogen or in frozen blocks embedded at optimal cutting temperatures for further RNA and histological analysis. In the atherogenic diet experiment, mice were treated with 15mg/kg of compound 1 after 3 weeks of diet. After 3.5 weeks of treatment, mice were sacrificed and livers were frozen in liquid nitrogen or in frozen blocks embedded at optimal cutting temperature. Plasma was collected from atherogenic diet fed mice and stored at-20℃for useThe analyzer and test strip (Roche) performed ALT and AST analyses. />

Statistical analysis

Statistical analysis WAs performed on the data using Excel software packages (Microsoft, redmond, WA) or GraphPad Prism6 (GraphPad Software inc., la Jolla, CA). The difference between the two groups was determined using a two-tailed Student t-test and Pearson and Spearman correlation coefficients. Data are given as mean ± SD and shown as error bars for all experiments. The difference was considered significant at P < 0.05. Reported data were obtained from at least 3 biological replicates.

Results

Effect of miR-122 on lipid metabolism in High Fat Diet (HFD) fed mice

Initially, we were interested in studying the function of MIR122 in HFD mouse livers that resulted in lipotoxicity, because in humans, MIR122 levels in NASH livers were significantly lower.

MIR122 reduces TG accumulation by targeting AGPAT1 and DGAT1 enzymes in the TG biosynthetic pathway. To test the effect of reducing MIR122 in the liver of HFD-fed mice, we administered antagoMIR122 to the liver by hydrodynamic tail vein injection, which blocks and degrades MIR122 in hepatocytes. Such injection resulted in a decrease in the level of mature MIR122 in the liver of Normal Diet (ND) fed mice and HFD mice. The levels of MIR122 precursors pri-and pre-MIR122 are also reduced. Furthermore, antagoMIR122 injection significantly reduced plasma levels of miR122 compared to unaffected miR-93. The antagoMIR122 injection also reduced MIR122 levels in distal White Adipose Tissue (WAT). In contrast to MIR122, decreased levels of miR-126 in HFD mice WAT compared to ND, decreased levels of antagoMIR122 resulted in a small, insignificant increase in miR-126 levels.

Decreased levels of mature MIR122 in WAT and muscle are the result of decreased secretion of MIR122 by hepatocytes, rather than decreased expression of MIR122 in non-liver tissues. The decrease in plasma MIR122 levels following antagoMIR122 injection was associated with an increase in liver fat droplet and total TG liver content and an increase in muscle TG levels. The reduced biochemical effects of liver MIR122 are manifested by a reduction in oxidation (reduced plasma levels of beta-hydroxybutyrate), as well as a reduction in liver cpt1α levels (carnitine palmitoyl transferase 1A), an important enzyme in the α -oxidative pathway and a reduction in plasma levels of Free Fatty Acids (FFA).

All of these are known signs of increased TG storage and decreased energy expenditure in tissues. Blocking of MIR122 by antagoMIR122 had a significantly increased overall effect on body weight of HFD mice. Liver weight and liver to body mass index also increased. Reducing the effects of MIR122 in the liver and systemic distant tissues results in increased liver lipids, reduced β -oxidation and energy expenditure, and has a systemic effect that mimics all of the features of the metabolic syndrome.

Expression of FGF21 (fibroblast growth factor 21), a known target of ROR alpha, was positively correlated with pre-MIR122 levels

In mice rorα regulated the expression of MIR122, which is mediated by FFAs8 (data not shown). To investigate the potential relevance to human metabolism and NASH, we initially studied the human NASH dataset (GSE 33814 and GSE89632, respectively). As described above, rorα is reduced in NASH patients. Furthermore, rora target genes (ArgI and CD 36) are reduced in these samples and their expression is positively correlated with rora. On the other hand, expression of genes involved in FFA biosynthesis pathway and associated with fatty liver (Fasn and Srebf 1) is inversely related to ROR alpha.

Similarly, MIR122 target genes (AldoA, ADAM17 and Agpat 1) are also inversely related to rora expression, and their levels in human liver increase with decreasing rora levels.

Importantly, expression of the known rora target FGF21 (fibroblast growth factor 21) correlated positively with the pre-MIR 122 level, indicating co-regulation (fig. 14).

Liver FGF21 was also up-regulated upon cold exposure. While not wishing to be bound by a particular theory, this may be due to an increase in rora levels in the cold. To evaluate this hypothesis, we transfected Huh7 human HCC cells with rora reporter plasmid expressing luciferase from MIR122 promoter containing one rora binding site.

When the temperature was reduced to 32 ℃, the expression of MIR122 increased (data not shown). When the ROR alpha consensus binding sequence in the MIR122 promoter was mutated, the increase in MIR122 expression disappeared. Furthermore, MIR122 levels and their activity on target genes increased in response to cold exposure of Huh7 cells.

To further investigate rora-MIR 122 circulation in humans, a human study (university of Hadassah hospital IRB approval No. HMO-0025-18) was performed in which MIR122 plasma levels were measured in humans undergoing large vessel cardiovascular surgery using a heart lung machine and systemic hypothermia (data not shown).

At reduced temperatures, a significant increase in plasma MIR122 levels was observed, which was positively correlated with an increase in plasma FFAs levels.

Effect of newly identified rora agonist compound 1 on mouse liver and whole body MIR122 levels

The results shown above verify the FFAs-MIR122-TGs metabolic cycle. Thus, increasing rora activity will result in increased liver MIR122 expression, which in turn results in increased FGF21 expression. For this reason, we have attempted to identify new rora agonists.

Targeted virtual screening was used to identify new rora agonists. A group of 300,000 pharmaceutical-like commercial compounds were docked and recorded into the crystal structure of the rora ligand binding domain complexed with cholesterol sulfate. The last 10 compounds were selected for activity testing. Induction of MIR122 promoter by this group of compounds was analyzed using luciferase promoter reporter plasmid (fig. 3A). Notably, compound 1 was most effective at inducing the MIR122 promoter, more effective than the commercially synthesized rora agonist SR 1078. SR1078 has the following structure:

a panel of cell lines was used to determine the toxicity profile of compound 1. Compound 1 was only moderately toxic (CC) in CEM and Huh-7 compared to cycloheximide (+control) ₅₀ 11.0 and 10.4 μm respectively) (data not shown). The chemical nature of the reagent was confirmed by resynthesis.

Compound 1 was further tested in a subsequent assay in which the rora DNA response element in the MIR122 promoter was mutated. In agreement with rorα specific activity, compound 1 showed no induction of the mutated MIR122 promoter, in contrast to the wild-type promoter. When Huh7 cells were exposed to compound 1 for 16 hours, there was no change in cellular MIR122 levels, but a large amount of MIR122 was secreted into the culture medium. Furthermore, there was no apparent toxicity to cells as measured by LDH release (data not shown). Importantly, treatment with compound 1 increased the activity of the MIR122 promoter in mice, and it was mediated by rorα, because MIR122 reporter plasmids carrying mutations at the rorα binding site exhibited reduced promoter activity compared to the wild-type (wt) promoter.

To further demonstrate that compound 1 is an effective inducer in mice, the levels of MIR122 in mice were monitored over time after a single administration of the compound. MIR122 levels increased in liver and plasma and correlated with increased levels of liver MIR122 precursors (pri-MIR 122 and pre-MIR 122), as well as decreased known target AldriA of MIR122 and increased known ROR alpha target gene G6Pase (Chauv C. Et al Control of gene expression by the retinoic acid-related orphan receptor alpha in HepG2 human hepatoma cells, PLoS One 6, e22545 (2011)).

After a single administration of compound 1, the level of mature MIR122 was significantly increased in WAT, muscle and heart tissue. Furthermore, in heart tissue, three MIR122 target genes were significantly down-regulated. These data demonstrate that compound 1 is a potent inducer of MIR122 in mice.

Compound 1 reduced weight and steatosis by increasing MIR122 expression in HFD-fed mice.

Human steatohepatitis is associated with reduced rora levels. Accordingly, there was a gradual decrease in liver rora levels in HFD fed mice over time. Interestingly, the addition of compound 1 agonist increased rora levels to their levels in normal diet fed mice.

According to our findings, compound 1 increased MIR122 levels, we further studied the mechanism of action of MIR122 and compound 1 on NAFLD to see if they are identical on the same pathway. Thus, we designed an experiment in which compound 1 was injected with the MIR122 inhibitor antagoMIR122 into mice with NAFLD.

Mice were fed 50% HFD and after 4 weeks, when fatty liver had formed, mice were treated with compound 1, antagoMIR122 or both compounds together for an additional 3 weeks. antagoMIR122 is administered once a week because Half-life extension, compound 1 was administered twice weekly because of plasma t _1/2 For 2.7 hours. At week 3, the week prior to starting the treatment, mice were initially weighed. Control mice (once weekly antagomiR-control and twice weekly DMSO diluted with saline) steadily increased in body weight, however, mice administered antagomiR122 increased most in body weight. In contrast, mice treated with rora agonist compound 1 showed a significant decrease in body weight. The body weight of the mice administered antagoMIR122 and compound 1 was fully restored to the body weight of the control animals. At the cessation of the experiment, the body weight of the antagoMIR 122-treated animals increased significantly, indicating that the reduction of MIR122 in the liver was associated with systemic effects, whereas administration of compound 1 significantly reduced the body weight of the mice. The liver weight of the mice was consistent with their body weight. The liver was further analyzed to assess lipotoxicity and liver lipid droplet and TG content was reduced in compound 1 treated mice and this reduction was completely eliminated in antagoMIR122 injected mice, indicating that the beneficial effect of compound 1 on steatosis was mediated by MIR122 activity.

We also measured the alternative marker of energy expenditure, β -hydroxybutyrate, and found that energy expenditure was reduced when MIR122 levels were reduced and increased when treated with compound 1. These effects are associated with reduced levels of mature MIR122 in liver, plasma and muscle tissue when antagoMIR122 is administered, while MIR122 levels are increased when compound 1 is administered, as seen in plasma and muscle.

Pri-MIR122 was not detected in muscle tissue, indicating that mature MIR122 was not expressed by the endogenous MIR122 promoter. The effect on muscle MIR122 levels is very similar to that seen in plasma and is likely through MIR122 secretion effects. The level of mature MIR122 in the liver did not increase after compound 1 administration, probably due to its secretion into the plasma, because MIR122 precursor RNAs pri-and pre-MIR122 in the liver increased significantly after compound 1 administration.

Importantly, after compound 1 treatment, MIR122 target gene Dgat1 was decreased in the liver, while rorα target gene FGF21 was increased (fig. 12).

MIR122 target genes AldoA and Agpat1 in muscle were also affected in a separate manner. Furthermore, in the presence of compound 1, muscle TG levels decreased. Following administration of compound 1, hepatic FGF21 mRNA levels correlated with pri-MIR122 levels, indicating co-modulation (fig. 13). These observations support our hypothesis that controlling MIR122 levels, either by lowering its liver levels by antagoMIR122, or by increasing its liver levels by compound 1, both of which may stimulate endogenous production of FGF 21. Thus, compound 1 and other rora agonists are useful for treating disorders associated with FGF21 levels, such as pancreatitis.

Compound 1 reduced steatosis by increasing MIR122 expression in HFD-fed mice.

Since compound 1 is a potent MIR122 activator, exhibiting beneficial biochemical effects, its effect on lipotoxicity and metabolism was determined. Compound 1 was administered to mice with established NAFLD (after HFD feeding) which resulted in increased liver and plasma maturation MIR122 levels, and increased MIR122 precursor and rorα target in the liver (fig. 14), similar to its effect on normal diet fed mice.

Compound 1 activates the anti-inflammatory and anti-fibrotic effects of the rora-MIR 122-triglyceride cycle

After we found that rora activator compound 1 showed significant metabolic benefits, we wanted to investigate its effect on liver inflammation and fibrosis. For this, we used a mouse atherogenic diet model. After 3 weeks of atherogenic diet, when liver inflammation and fibrosis has progressed, we began to treat with compound 1 for an additional 3.5 weeks, 3 injections per week. Animals were then evaluated for their effect on compound 1 in a number of procedures. Compound 1 improved liver enzymes, significantly reduced AST and ALT levels. After administration of compound 1, the level of mature MIR122 in liver and plasma increased. H & E, CD3 and F4/80 staining showed that compound 1 significantly improved liver inflammation. The improvement in inflammation was associated with a significant reduction in liver fibrosis as assessed by both Masson trichrome (m.t.) and smooth muscle actin (αsma) staining. NK cells are known to target activated hepatic stellate cells. Regression of fibrosis was associated with a significant reduction of NK cells in the liver of compound 1 treated mice.

As shown in fig. 12, the effect of rorα activator compound 1 on fibrosis driving genes is also apparent. These results strongly support the concept that rora agonists might reduce liver inflammation and fibrosis by upregulating MIR 122. While not wishing to be bound by a particular theory, it is believed that ROR alpha agonists may treat pancreatitis by reducing and/or reversing fibrosis due to the high degree of fibrosis present in pancreatitis patients.

Discussion of the invention

In this report, we show that activating rorα can bring significant health benefits. This rora activity is mediated by inducing liver MIR122 levels, although additional rora activity may contribute to these beneficial effects. MIR122 in turn increases expression of FGF 21. Rora activation also results in increased secretion of liver MIR122 into plasma, resulting in increased levels thereof in distant tissues such as WAT, muscle and myocardium, where MIR122 also affects its target genes. Thus, activating rorα has an effect on both the liver and the whole body.

We identified a synthetic rora activator in many compounds, compound 1, through which there was a strong enhancement of MIR122 promoter activity. By testing the effect of compound 1 in several mouse models, we found that this compound has a significant beneficial effect on increasing FGF21 levels and is therefore useful in the treatment of pancreatitis.

The treatment of pancreatitis is highly preferred because there is currently no drug approved for pancreatitis other than palliative treatment. Thus, pancreatitis patients still have no treatment options. Many compounds are in the drug development stage, some showing interesting prospects, and some failing to reach an important endpoint. We decided to investigate the potential therapeutic effect of MIR122 because it has hormone-like properties. That is, MIR122, like many other micrornas, is produced in one tissue (liver) and then secreted into the blood from where it reaches the distal tissue. We have demonstrated that MIR122 exerts its antilipemic effects in the liver and distal tissues.

Hepatocytes produce large numbers of MIR122, up to 250,000 copies per cell. The potent distal activity of MIR122 is associated with its high yield and secretion to produce high plasma levels. Thus, induction of high productivity of MIR122 can be converted into an effective therapeutic compound. However, we have not developed a system for synthesizing MIR122 (MIR-MIR 122) as a drug and injected into NASH patients for many years, we have preferably developed a small drug that induces endogenous expression of MIR122 in the liver and subsequently induces endogenous expression of FGF21, and can be administered to patients for a long period of time.

In this report, we demonstrate that compound 1 specifically activates rorα, thereby increasing the level of MIR122 in the liver and resulting in increased FGF21 expression. Based on our findings, ROR alpha activators are considered a promising compound to be developed and evaluated for its clinically beneficial effects in pancreatitis patients.

Example 13

Rora activation can reverse pancreatitis by FGF21 activation

Pancreatitis is a common debilitating clinical condition that results in high morbidity and mortality. There is no specific therapy for this severe clinical condition. Pancreatitis is treated very limitedly and is usually only supportive. Pancreatitis begins with the activation of digestive enzymes in the pancreas, which results in tissue damage and inflammation. Common causes of pancreatitis include alcoholism, hyperlipidemia, and gall stones moving out of the biliary tract. Pancreatitis is also iatrogenic, occurring in 5-10% of patients receiving Endoscopic Retrograde Cholangiopancreatography (ERCP). Overall, pancreatitis is an unmet therapeutic need.

Fibroblast growth factor 21 (FGF 21) is a hormone secreted by the liver in response to various metabolic stresses. FGF21 is expressed in the exocrine pancreas to stimulate digestive enzyme secretion. FGF21 KO mice are particularly prone to pancreatitis. Overexpression of FGF21 provides protection against pancreatitis. Prophylactic administration of FGF21 reduced fibrosis in a mouse model of pancreatitis. Loss of FGF21 is a driving factor for pancreatitis. FGF21 was used to therapeutically reverse pre-existing pancreatitis.

We have shown that FGF21 can be activated upon treatment of cells with ROR alpha agonist compound 1, as shown directly and indirectly below (see FIGS. 11-15)). Thus, we have unique therapeutic potential against pancreatitis for compounds that activate rorα.

Example 15

Pancreatitis screening assay

To assess the ability of the RORA agonist compounds described herein to treat, prevent, reduce the susceptibility to, reduce the severity of, or delay progression of pancreatitis, it would be beneficial to use an animal model of pancreatitis.

While chronic alcoholism is the primary cause of chronic pancreatitis, many other causes (including toxins, obstructive lesions, and genetic diseases) can also lead to chronic pancreatitis.

In a traditional acute pancreatitis model, recurrent episodes of acute pancreatitis can lead to fibrosis. An example of such a model is the induction of chronic pancreatitis by repeated injections of rana peptide. The Sensory Acute Pancreatitis Event (SAPE) model suggests that an initiating event (e.g., an acute pancreatitis episode) activates the immune system, allowing risk factors to drive pro-fibrotic, anti-inflammatory pathways, leading to chronic pancreatitis. The ethanol sensitization model (e.g., ethanol/lipopolysaccharide model) meets this assumption. Both pancreatitis models resulted in similar severity of the final pancreatic injury.

All animal models of chronic pancreatitis, except autoimmune models, have the same histological endpoints (i.e., fibrosis, pancreatic duct abnormalities, and cellular changes), whether caused by chemical exposure, dietary changes, infectious agents, genetic modifications, or mechanical disorders. Examples of animals that can be used in these models include cats, dogs, ferrets, mice, rats, pigs, rabbits, and zebra fish.

These include some or all of the following features chronic inflammation, stellate cell proliferation/activation, acinar cell shedding, ductal dilation, intraductal calcification and nerve enlargement.

An animal model comprises repeated intraperitoneal injections of cholecystokinin (CCK) analogues, rana peptide, which bind to Lipopolysaccharide (LPS), chronic ethanol, cyclosporin A or dibutyltin Dichloride (DBTC). Another animal model involves chronic administration of ethanol and LPS. Another model involved administration of relatively large doses of arginine. Yet another model involves administration of a choline-deficient, supplemental ethionine (CDE) diet. Another model involved administration of trinitrobenzenesulfonic acid (TNBS).

Among them, the repeated administration of rana peptide, sometimes in the presence of a sensitizer, is the most common model of chronic pancreatitis. These and other toxic compounds can be administered systemically, intraperitoneally, or by retrograde infusion into the pancreatic duct.

In a widely used animal model of acute pancreatitis, treatment is performed with a supraphysiological dose of rana peptide. At low doses, rana peptide provides physiological stimulation of CCK receptors and enhances secretion of acinar cells. However, mild to moderate acute interstitial pancreatitis developed with high doses of rana peptide (10-100 Xphysiological doses; 20-50. Mu.g/kg, intravenous or intraperitoneal). The rana peptide model of acute pancreatitis is characterized by abnormal zymogen activation, secretion inhibition, increased inflammation and cell damage in acinar cells. However, in this model of pancreatitis, the exocrine pancreatic structure and function was restored within 24 to 48 hours.

The rana peptide model of chronic pancreatitis requires repeated injections of rana peptide over time, and is the most common and repeatable model of chronic pancreatitis. There are many regimens that differ in the dose, interval and duration of the rana peptide injection (Feng et al, int J Biol Sci 8:249-257,2012; neuschwander-Tetri et al, dig DisSci 45:665-674,2000; yadav et al, am J Gastroenterol 106:2192-2199,2011), however any of these regimens may be used. The rana peptide model produced morphological findings consistent with human chronic pancreatitis, including fibrosis, chronic inflammation, atrophy, acinar differentiation and transfer into ductal-like cells and ductal dilation.

The rana peptide model of chronic pancreatitis is the basis for studying the sensitization of other drugs. Lipopolysaccharide (LPS) is a bacterial endotoxin, a particularly relevant substance, since long-term drinking results in increased intestinal permeability, susceptibility to bacterial translocation and increased serum LPS levels. LPS has been shown to activate pancreatic stellate cells and stimulate inflammatory cytokines by activating toll-like receptor 4 (TLR 4) and nuclear factor κb (nfκb). The addition of LPS to the model of repeated injection of rana peptide accelerated disease progression and worsened its severity as measured by acinar cell atrophy, fibrosis and tubular complex progression (Ohashi et al Am J Physiol Gastrointest Liver Physiol 290:290G 772-G781,2006.).

Cyclosporin a (CsA) is also used as a sensitizer for rana peptide-induced chronic pancreatitis. In this model, rats received only two doses of rana peptide for intraperitoneal injection in 15 days of intraperitoneal CsA treatment. Rats treated with rana grahami peptide alone recovered completely from acute rana grahami peptide pancreatitis, whereas rats co-treated with cyclosporin exhibited chronic pancreatitis with atrophy, mononuclear inflammatory infiltrate and enhanced collagen deposition (Vaquero et al, gut 45:269-277,1999).

Feeding a choline-deficient, ethionine (CDE) -supplemented diet induces acute hemorrhagic pancreatitis in mice (Gilliland L and M.L. Steer, am J Physiol 239: G418-G426,1980). The mechanism by which CDE causes pancreatic injury is not yet known. Intermittent chronic administration of CDE diet for longer than 24 weeks resulted in histological changes consistent with chronic pancreatitis, including acinar atrophy, fibrosis, and tubular complex formation. Furthermore, increased expression of EGFR, SPINK3 and TGF-alpha was observed in this model, all of which are associated with pathogenesis from chronic pancreatitis to pancreatic cancer. However, even after CDE feeding for 54 weeks, no malignant lesions formed.

L-arginine, an essential amino acid, has been shown to cause severe necrotizing acute pancreatitis in animal models by intraperitoneal administration at high doses (Mizunum et al, J Nutr 114:467-471, 1984). Repeated injections over several weeks resulted in necrosis in rats with lower doses of L-arginine with severe acute disease, followed by chronic inflammation and fibrosis with impaired glucose tolerance.

Intravenous or intraperitoneal injection of dibutyltin Dichloride (DBTC), a compound used to produce polyvinyl chloride, causes chronic biliary obstruction by direct toxicity to acinar cells and by the formation of obstructive emboli in the distal common bile duct, leading to acute interstitial pancreatitis (Merkord J and Hennighausen G, exp Pathol36:59-62,1989). When DBTC is administered by repeated injections, chronic inflammation and fibrosis appear in rats. However, this model is not highly reproducible, as only one third of animals show histological changes consistent with chronic pancreatitis.

Reverse infusion of toxic substances

Several models involving retrograde infusion of toxic substances have been tried. These models only deliver toxins to the pancreas, unlike the models described above that require systemic administration of toxins. Infusion of trinitrobenzenesulfonic acid into pancreatic ducts resulted in acute necrotizing pancreatitis at 48 hours and fibrosis, inflammation and atrophy consistent with chronic pancreatitis at a later time point (Puig-Divi et al, pancrees 13:417-424,1996). Retrograde infusion of bile acids provides a considerable model for studying acute pancreatitis, as cholelithiasis obstruction is a common cause of acute pancreatitis (Perides et al, gastroenterology 138:715-725,2010). This approach is believed to trigger pancreatitis through direct toxic effects on acinar cells mediated by the bile acid receptor Gpbar 1.

Any of these models can be used to judge the effectiveness of the compounds described herein, alone or in combination with other active agents, in treating pancreatitis, preventing pancreatitis, reducing the susceptibility of pancreatitis, reducing the severity of pancreatitis, or slowing the progression of pancreatitis.

For example, one or more control animals may be administered rana peptide according to one of the above regimens, and one or more test animals are administered rana peptide, also treated with a RORA agonist compound described herein. The progression of pancreatitis can be monitored in test and control animals. Thus, pancreatitis prevention or reduced susceptibility, reduced severity, or delayed progression may be monitored.

Alternatively, one can wait until those animals administered a compound that causes the development of pancreatitis actually develop pancreatitis, and then evaluate the effectiveness of the RORA agonist compounds described herein in treating, lessening the severity of, or slowing the progression of pancreatitis.

Agents and RORA agonistsIs a combination of (a)

There are (at least) seven classes of drugs associated with acute pancreatitis, statins, ACE inhibitors, oral contraceptives/Hormone Replacement Therapy (HRT), diuretics, antiretroviral therapy, valproic acid and oral hypoglycemic agents. Although the mechanism by which these drugs cause pancreatitis is not yet defined, it is believed that statins have a direct toxic effect on the pancreas, or a toxic effect through the long-term accumulation of toxic metabolites. At the same time, ACE inhibitors cause pancreatic angioedema through the accumulation of bradykinin. Contraceptives and HRT lead to pancreatic arterial thrombosis (hypertriglyceridemia) through fat accumulation. Diuretics such as furosemide have a direct toxic effect on the pancreas. Meanwhile, thiazide diuretics cause hypertriglyceridemia and hypercalcemia, the latter being a risk factor for pancreatic stones.

HIV infection itself can lead to a person more susceptible to pancreatitis, while antiretroviral drugs can lead to metabolic disorders such as hyperglycemia and hypercholesterolemia, which can lead to pancreatitis.

Valproic acid may have a direct toxic effect on the pancreas. There are various oral hypoglycemic agents, such as metformin, which can cause pancreatitis. Atypical antipsychotics such as clozapine, risperidone and olanzapine can also cause pancreatitis.

When combined with one of the above-described agents that may cause pancreatitis, any of the models described above may be used to determine the effectiveness of a compound described herein in treating, preventing, reducing the susceptibility to, lessening the severity of, or slowing the progression of pancreatitis caused by such other agents.

For example, statin, ACE inhibitor, oral contraceptive/Hormone Replacement Therapy (HRT), diuretics, antiretroviral therapy, valproic acid or oral hypoglycemic agents (such as metformin) may optionally be administered to one or more control animals at dosages above normal dosages to accelerate the development of pancreatitis, and combinations of such agents with a RORA agonist may be co-administered to the treated animals to determine the effectiveness of the RORA agonist in preventing, reducing the susceptibility to, lessening the severity of, or delaying the development of pancreatitis caused by these other agents. Thus, prevention or reduced susceptibility, reduced severity or delayed progression of pancreatitis may be monitored.

Pharmaceutical compositions comprising a RORA agonist and a compound selected from the group consisting of statins, ACE inhibitors, oral contraceptives/Hormone Replacement Therapy (HRT), diuretics, antiretroviral therapy, valproic acid and oral hypoglycemic agents (e.g. metformin) are within the scope of the embodiments described herein.

Example 16

Animal model for apoplexy

Animal models of stroke may be used to evaluate the effectiveness of the RORA compounds described herein in treating, preventing, reducing the susceptibility to, reducing the severity of, or slowing the progression of stroke.

Animal models of stroke are well known and have been used to test reanalyzing, neuroprotection, nerve regeneration or anti-inflammatory drugs in preclinical settings.

One such animal model includes distal occlusion of the rat Middle Cerebral Artery (MCA). Different techniques and methods have also been developed to induce local and global ischemia in the brain. Specific models mimic different types of stroke, ischemia, and global ischemia.

Cerebral ischemia models can be classified into ischemia models and global ischemia models. Focal ischemia is characterized by a decrease in cerebral blood flow in a specific area of the brain, whereas in global ischemia, a decrease in blood flow affects the entire brain or forebrain (Traystman rj. Animal models of focal and global cerebral ischemia. Ilar journ/National Research Council, institute of Laboratory Animal resources.2003;44 (2): 85-95).

Strokes caused by acute cerebrovascular occlusion can be replicated by different techniques, namely by mechanical occlusion of the proximal middle cerebral artery (pMCAo) (large vessel occlusion) or distal MCA (dwcao) (small vessel occlusion), or by thrombotic occlusion by injection of blood clots or thrombin into MCA, or by light plug therapy after intravenous injection of manalar red.

The pMCAo model is commonly used in stroke studies. pMCAo is usually induced by direct mechanical occlusion, most commonly by inserting a silicon coated nylon suture into the internal carotid artery, followed by advancement to the Willis ring to occlude the MCA at its origin. The severity of ischemic injury can be simulated by leaving the suture temporarily in place for a variable period of time (typically between 30-120 minutes) before removing the suture to allow tissue reperfusion. In the case of permanent pMCAo, the suture remains in place and reperfusion is not allowed. Short duration pMCAo resulted in selective neuronal death in the injured striatum, expression of heat shock proteins, immediate early gene expression, and induction of apoptotic signaling pathways in the epithelial layer. Conversely, prolonged occlusion can lead to cerebral infarction involving the striatum and cortex, and may be associated with death of some animals in the event of oedema formation.

Human stroke is most often caused by cerebral thromboembolism. Thus, a number of animal models have been developed that closely mimic the embolic occlusion of cerebral vessels. Embolic stroke can be induced in animals by injecting large size synthetic microspheres (300-400 μm in diameter) or small size microspheres (less than 50 μm) into the internal carotid artery. In the first case, a large area of infarction similar to that produced by MCA permanent occlusion is induced. In the latter case, minor multiple infarctions may occur (Gerriets et al, J Neurosci methods 2003;122 (2): 201-11; miyake et al, stroke 1993;24 (3): 415-20).

These models can be used to evaluate the effectiveness of a RORA agonist described herein in treating stroke, preventing stroke, reducing susceptibility to stroke, reducing the severity of stroke, or slowing the progression of stroke. To test the effect of treatment or delay the progression of stroke, these compounds may be administered after mechanically inducing stroke. To test for preventing stroke, reducing susceptibility to stroke, or reducing severity of stroke, the compound may be administered prior to mechanically inducing stroke. When a combination of treated animals and control animals is used, the effectiveness of the compound can be compared to the control.

A different type of model was used to study thrombolytic therapy. Autologous blood clots injected directly into the internal carotid artery were used to induce vascular occlusion (Kilic et al, neuroreport.1998;9 (13): 2967-70). Animal models of this type can be used to evaluate combination therapies using the RORA agonist compounds described herein in combination with agents that dissolve blood clots (e.g., tPA). Compositions comprising a RORA agonist compound and a compound that solubilizes or removes blood clots are another embodiment of the invention described herein.

Example 17

Animal model for sarcopenia

About 40-50% of the population over 80 years of age suffers from sarcopenia, which makes this a major clinical disorder in elderly and a key challenge in healthy aging. The hallmark symptom of sarcopenia is a loss of muscle mass and strength, and patients with sarcopenia may have worse clinical outcome and higher mortality than healthy individuals.

Animal models designed for the study of sarcopenia include hindlimb unloading, denervation, and activity limitation by using plaster or wire strategies and using geriatric rodents.

Senior rodents are commonly used in animal models of sarcopenia. For example, female C57BL/6J mice developed sarcopenia at 24 months, a significant loss of quadriceps femoris muscle mass, which was more pronounced at 27 to 29 months, when neuromuscular junction (NMJ) morphologies were altered by denervation and myofiber (Shavelakadze T and groups M, bioessays.2006;28:994-1009; chai et al, PLoS one.2011; 6:e28090). Gait characteristics of the aged mice were also changed. Older mice (24 months old) exhibited significantly reduced stride frequency, increased stride time variability, and altered footstep patterns compared to young mice (3 months old). The aged rat model also showed a muscle reduction pattern similar to that of the aged mouse model.

Because high caloric intake is known to accelerate the development of sarcopenia, some animal studies may involve providing animals with a high fat diet.

Other animal models include induction of muscle atrophy, for example, by use of hindlimb movement limitation methods. Including low head suspension with single hind limb support, tail suspension with adhesive tape traction, whole body suspension with hind limb bearing, and hind limb plaster tail suspension. These "weight loss" models can lead to muscle loss.

Denervation is a common phenomenon of senile neuromuscular junctions (NMJs). Some common rodent models use tibial or sciatic nerve transection to induce denervation. The tibial nerve is a mixed motor sensory peripheral nerve in the rodent hindlimb and is one of the 3 terminal branches of the sciatic nerve. Transection of the tibial nerve will denervate the gastrocnemius, soleus and plantar muscles.

If hindlimb function assessment is required, walking trajectory analysis may be performed at different time intervals. This involves immersing the feet of the animal in the ink and allowing the animal to walk through a paper-backed enclosure. Features of the footprint can be reliably measured and scored to indicate the extent of neuromuscular disability and gait impairment, as the footprint features reflect functional muscle groups.

These animal models can be used to assess the ability of the RORA agonist compounds described herein in treating, preventing, reducing the susceptibility of, reducing the severity of, or slowing the progression of sarcopenia, especially when used in combination with control animals that have not received any treatment.

Example 18

Traumatic brain injury animal model

Animal models of Traumatic Brain Injury (TBI) are used to determine potential neuroprotective therapies for developing and adult brains.

Traumatic brain injury is a complex process consisting of four stages overlapping each other, including primary injury, evolution of primary injury, secondary or additional injury, and regeneration. Primary injury to the brain can be induced by a variety of mechanisms. One mechanism involves direct brain bruising from the skull. Another mechanism involves brain bruises caused by motion striking the rough inner surface of the skull, and/or indirect brain bruises opposite the striking side. Another mechanism involves shearing and stretching of brain tissue caused by movement of brain structures relative to the skull and each other. Another mechanism involves vascular responses to impulses, including subdural hematomas resulting from rupture of bridging vessels between the brain and dura mater, decreased blood flow due to increased intracranial pressure or infarction, and cerebral edema resulting from increased permeability of cerebral blood vessels.

Diffuse axonal injury is considered one of the major consequences of blunt head trauma; it is characterized by morphological and functional damage to axons throughout the brain and brain stem and leads to diffuse degeneration of white matter of the brain. Secondary injury mechanisms include complex biochemical and physiological processes that are initiated by primary injury and manifest themselves over hours to days.

Animal models attempt to replicate certain pathological components or stages of clinical trauma in experimental animals, and then allow one to evaluate the putative treatment. Rodent models are commonly used in nerve trauma studies. Their relatively small size allows for repeated measurements of morphological, biochemical, cellular and behavioral parameters requiring a relatively large number of animals. Animals typically suffer from two major types of experimental brain injury, namely accelerated concussion and concussion.

Mechanical forces cause dynamic or static brain damage, depending on its amplitude, duration, speed and acceleration. The mechanical forces in the static model have a determined amplitude and duration, but no velocity or acceleration. In essence, static models are typically concerned with the morphological and functional processes involved in the injury. One example of a static injury model includes squeezing the cranial nerve with forceps for a defined period of time.

Dynamic brain injury can be induced by applying mechanical forces with well-characterized amplitudes, durations, velocities, and/or accelerations. Dynamic brain injury can be further subdivided into direct injury and indirect injury. In the case of indirect dynamic brain injury, mechanical forces are typically directed throughout the body, with the kinetic energy of the oscillating pressure wave passing through the body, exerting an effect on the brain tissue.

Head penetration injuries and other direct brain deformation models are caused by impact energy transmitted to the brain parenchyma through the skull penetrated by the projectile or craniotomy. Drug treatments using these models to assess TBI have been established (Faden et al, science,1989May 19;244 (4906): 798-800).

The lateral fluid impact model provides a lesion that replicates the clinical bruise without skull fracture and shows a direct relationship between most pathological changes and the severity of the lesion. It is widely used in mechanism studies and drug screening in nerve trauma studies.

Other models use controlled cortical impact to cause traumatic brain injury in rats (Dixon et al, JNEurosci methods 1991Oct;39 (3): 253-62).

These and other animal models can be used to assess the effectiveness of the RORA agonist compounds described herein in treating TBI, reducing its severity or duration, or slowing its progression, particularly when comparisons can be made between treated animals and control animals. The compound may be administered prior to, concurrently with, or after induction of brain injury, optionally in combination with other active agents for the treatment of traumatic brain injury. Pharmaceutical compositions comprising a RORA agonist compound and an additional active agent are within the scope of the invention described herein.

Example 19

Pancreatitis is an example of pancreatic inflammation. The most common causes of pancreatitis include gallstones (40%), alcoholism (33%), spontaneity (15-25%) and post Endoscopic Retrograde Cholangiopancreatography (ERCP) (5-10%). Pancreatitis treatment is limited and is often supportive. The total mortality rate for acute pancreatitis is 10-15%. Thus, there is an urgent need to find a treatment for pancreatitis.

According to recent papers, FGF21 may be used as a treatment for pancreatitis (Hernandez, G. Et al, pancreatitis is an FGF-deficient state that is corrected by replacement treatment.science Translational Medicine, (2020)). ROR- α is one of the transcription factors regulating FGF21 (Luo, Y. Et al Oncogenic KRAS Reduces Expression of FGF21 in Acinar Cells to Promote Pancreatic Tumorigenesis in Mice on a High-Fat diet. Gastroenterology 157,1413-1428.E11 (2019)).

ROR-a agonists such as RS2982 and the compounds described herein may be used in models that induce pancreatitis, for example in two different mouse models as described herein, to assess their therapeutic effect on pancreatitis.

In the first model, rana peptide induced pancreatitis (CIP) (see, e.g., hyun, J.J. & Lee, H.S. experimental models of clinical endoscope 47,212-216 (2014)), mice of 6-10 weeks of age were intraperitoneally injected seven times per hour with rana peptide (50 ug/kg). The control group was injected with physiological saline. 24 hours after the first injection, mice were injected with RS2982 (2.5-25 mg/kg) or DMSO and examined for pancreatitis one day later.

In the second model, alcohol-induced pancreatitis (AIP), 6-10 week old mice were injected intraperitoneally with ethanol (1.3 g/kg) and POA (150 mg/kg) twice within 1 hour (Huang, W.et al, fatty acid ethyl ester synthase inhibition ameliorates ethanol-reduced Ca2+ -dependent mitochondrial dysfunction and acute pancreitis. Gut 63,1313-1324 (2014)). 24 hours after the first injection, mice were injected with RS2982 (2.5-25 mg/kg) or DMSO and examined for pancreatitis one day later.

Using one of these models, RT-qPCR analysis of FFG21 mRNA expression was performed in 266-6 murine acinar cells after 6 hours of DMSO/different doses of SR1078 (commercial ROR-a agonist). Statistical significance was calculated relative to DMSO and determined by Student t-test (two-tailed). Values in the graph are mean ± SD. * p <0.05, < p <0.01, < p <0.001, < p <0.0001.

The data shown in fig. 16 demonstrate that SR1078 increases the expression of Fgf21 in acinar cells. The best effect was observed at a concentration of 10 micromolar (μm), with decreasing effect as the dose increased to 20 and then to 40 micromolar (μm).

Example 20 analysis to identify potent ROR alpha (RORA) agonists

The ability of a compound to bind to the RORA receptor, to have agonist activity when bound to the receptor, and to not cross the blood brain barrier and/or to not bind to GABA receptors are important considerations for compounds useful in the methods described herein.

Agonists, inverse agonists, antagonists bind in the same binding pocket of the RORA LBD (ligand binding domain). FIG. 17 shows the binding domain, as well as compounds embedded within the domain.

Many compounds are known to be agonists and inverse agonists of the RORA receptor:

RORA agonists

Cholesterol

RORA inverse agonists

Therefore, it is important to identify not only compounds that bind the RORA receptor with relatively high affinity, but also compounds that are agonists rather than antagonists, inverse agonists, etc.

Rora agonists are useful for increasing liver microRNA122 (MIR 122) expression levels and are useful in the treatment of pancreatitis, sarcopenia, stroke, glioblastoma, and traumatic brain injury, as well as fatty liver diseases, including fatty liver (NASH) and related diseases. Increased expression of RORA in mouse liver increases expression of MIR122 and reduces lipotoxicity.

The assay disclosed by Chai et al (Chai et al, "Agonist of RORA Attenuates Nonalcoholic Fatty Liver Progression in Mice via Up-regulation of MicroRNA122," gastroenterology 2020;159 (3): 999-1014.e9) is one example of an assay that can be used to identify RORA agonists. The assay is used to screen a library of compounds and identify ROR alpha agonists.

Materials and methods

Cell culture

The human hepatocellular carcinoma cell line Huh7 was cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Thermo Fisher Scientific, waltham, MA). The cells were incubated at 37℃in the presence of 5% CO ₂ Except for the experiments in which the cells were placed at 32℃as indicated herein.

Plasmid(s)

Human MIR122 promoter fragments spanning the-900 base pair region relative to the transcription initiation site and mutating the RORA binding sites (plasmids pMIR122-900 and pMIR122-RORAmut, respectively) were generated as described previously.

Transfection

For the luciferase assay, cells grown on 24 well plates were co-transfected with a luciferase reporter plasmid (50 ng) and 1ng of Renilla luciferase vector (PRL, promega) using liposome LTX (Invitrogen, waltham, mass.) transfection reagent. For all experiments, transfection was performed using serum-free medium (Opti-MEM; cat #31985070;Thermo Scientific).

Luciferase Activity assay

Following transfection, cells were lysed with passive lysis buffer (catalogue number E1941; promega), shaken at room temperature for 20min, and then transferred to a suitable 96-well plate. Firefly and Renilla luciferase activities were evaluated on a photometer Mithres 2000 (Centro XZ, LB960, berthold Technologies, bad Wildbad, germany) using a dual luciferase reporter assay system (Cat#E1910; promega). Normalizing the luciferase activity to a renilla luciferase activity. Readings were taken in triplicate.

Virtual screening can be targeted to identify new RORA agonists. A library of commercially available compounds can be docked and recorded into a crystal structure model of the RORA ligand binding domain complexed with cholesterol sulfate.

Once the lead compounds are identified, they can be screened for activity assays, for example, by analyzing their induction of the MIR122 promoter using the luciferase promoter reporter plasmid described above. As shown in fig. 18A and B, compounds that bind to the appropriate miR-122 promoter site, once bound, exhibit agonist activity, will induce luciferase activity (fig. 18A), and are thus identified as RORA agonists. If the compound does not bind to the appropriate site, and/or does not induce luciferase activity (fig. 18B), it will not be identified as a RORA agonist. Thus, the screening assay can be used to identify potential RORA agonists.

Using this screening assay, compound 68 was identified as a potent MIR122 promoter, more potent than the commercially synthesized RORA agonist SR 1078. FIG. 19 shows the results of screening assays for wild-type and mutant RORA at concentrations of 0.5, 1 and 5. Mu.M.

Compound 68 has the formula:

/>

since substitution on one or more aryl rings or changes in the size of the heterocycle are not expected to significantly alter the affinity of the compound for the RORA receptor, or alter the activity, or alter the ability of the compound to cross the blood brain barrier and/or bind to GABA receptor, the general formula of these compounds is shown below, and these compounds are within the scope of the compounds described herein:

And pharmaceutically acceptable salts and prodrugs thereof, wherein R ² And u is as defined above for formula A, except that u may also be 0, and n is 0, 1 or 2.

Because compound 68 has a benzodiazepine core:

other benzodiazepines also potentially have RORA activity and can be screened using the assay described above for this activity.

Representative Benzodiazepines (BDZs) are known to have pharmacological activity, although binding to the RORA receptor is not known, as follows:

BDZ drugs are commonly used to treat anxiety, seizures, and insomnia and provide sedation, their mode of activity in the central nervous system is generally associated with their binding to GABA receptors (e.g., GABA-A receptors) in the brain. In order to bind to the GABA-A receptor, the drug must cross the Blood Brain Barrier (BBB).

In certain embodiments, the compounds cross the blood brain barrier and also exhibit RORA agonist activity, and in these embodiments, they are useful for treating traumatic brain injury. In these embodiments, the effect of the compounds on the GABA-A receptor must also be considered, but these compounds are effective in such treatment when it is acceptable or desirable to utilize these effects while treating a patient with traumatic brain injury.

In other embodiments, the compound does not cross the blood brain barrier, or does not cross the blood brain barrier at appreciable concentrations, and thus has minimal or no effect on GABA receptors in the brain. Thus, they will not have the effects traditionally associated with BDZ drugs, i.e. will not cause sedation, and may be useful in the treatment of diseases other than traumatic brain injury as discussed herein.

There are predictive models for determining whether a compound has CNS activity, such as the assays disclosed in Gourdeau, h., mcAlpine, j.b., range, m. et al, identification, characterization and potent antitumor activity of ECO-4601,a novel peripheral benzodiazepine receptor ligand.Cancer Chemother Pharmacol 61,911-921 (2008). Using this assay, the compound diazepam was predicted to have no CNS activity, which was later demonstrated experimentally. Thus, this assay is a reasonable predictor of CNS activity for BDZ and other compounds described herein. Briefly, this assay involves screening compounds for their ability to bind to two different receptors, the peripheral and central benzodiazepine receptors. When the compound binds to the peripheral benzodiazepine receptor but not to the central benzodiazepine receptor, or the peripheral benzodiazepine receptor exhibits a significantly higher binding affinity (i.e., a ratio of 5/1 or higher, 10/1 or higher, 20/1 or higher, or most preferably 50/1 or higher) than the central benzodiazepine receptor, the compound is not expected to exhibit significant CNS side effects even though they cross the blood brain barrier. Assays for screening compounds for binding affinity to various receptors (e.g., peripheral and central benzodiazepine receptors) are well known to those of skill in the art and need not be discussed in greater detail herein.

Using this assay, a series of BDZ compounds were screened and the results are shown in the table below.

Drug molecules	CNS	QPlogBB	QPlogS
				Compound 68	-1	-0.991	-6.808
Olanzapine	2	0.758	-4.230
				Chlorine nitrogenFlat plate	2	0.895	-4.240
Luosaping (a medicine for treating lobar disease)	2	0.991	-3.870
				Pirenzepine	1	0.158	-1.003
Diazepam mould	-2	-2.097	-7.326
				Sintamil	0	-0.854	-1.456

In the above table, the "CNS" column shows predicted central nervous system activity ranging from-2 (inactive) to +2 (active). The column "Qlog BB" shows the predicted brain/blood partition coefficient. The ideal qLogBB range for drug avoidance BBB is-3.0-1.2. The more positive this value, the more likely the compound is to pass the BBB. Qlog S column shows predicted water solubility, log S, where S is in mol/dm ^-3 Represents the concentration of solute in the saturated solution in equilibrium with the crystalline solid. The desirable range of Qlog for the compounds described herein is-6.5 to 0.5, the more negative this value, the less soluble the compound. Using these predictive models, it is believed that compound 68 is unlikely to be CNS active.

These models can also be used to screen for other benzodiazepine derivatives, including the compounds described herein.

Bromodomain inhibitors (BDZ derivatives)

The compounds (+) -JQ1, (+) -MS417 and I-BET are benzodiazepine derivatives, also known as bromodomain inhibitors, and are non-CNS active (Smith et al, "Privileged Diazepine Compounds and Their Emergence as Bromodomain Inhibitors," Chemistry & Biology, volume 21,Issue 5,Pages 573-583 (2014)). Their structures are shown below:

As with the compounds in the table above, the likelihood of these three compounds crossing the blood brain barrier was also assessed. The results are shown in the following table:

based on this information, it is predicted that the central nervous system activity of these and other bromodomain inhibitors is lower than other BDZ drugs in the list.

Traditional BDZ drugs exhibit CNS activity through binding to the GABA-A receptor. To further determine whether these compounds have CNS activity across the blood brain barrier, a study may be conducted to determine their binding to the GABA-A receptor.

Molecular docking may be performed with the pdb structure of the GABA-A receptor, for example, using one or more protein database structures of the GABA-A receptor, such as 6X3X (subtype α1- β2- γ2 of human GABAA receptor complexed with GABa plus diazepam), 6X3U (subtype α1- β2- γ2 of human GABAA receptor complexed with GABa plus flumazenil). Representative computer molecular docking studies were performed on compounds 68 having two pdb structures of the GABA-A receptor (protein database (pdb) id 6X3X and 6X 3U). Based on the docking score, compound 68 has a lower binding affinity to the GABA-A receptor than the known BDZ drugs and is therefore expected to show little CNS activity even though it crosses the blood brain barrier.

Virtual screening has proven to be a very successful approach for finding ligand hits and assisting in lead optimization in structure-based drug discovery programs. By docking a large library of compounds into one or more high resolution structures of a target receptor, it is often necessary to experimentally screen fewer compounds to identify expected lead compound optimization candidates.

In addition to identifying small molecules that may bind well to protein targets, the docking method is also used in a variety of situations, such as gesture prediction of polypeptides and macrocycles, prediction of the geometry of protein-ligand complexes, and preparation of homologous sequences for binding affinity prediction using free energy perturbation or MM-GBSA methods. The method includes average gas phase energy (MM) and solvation free energy as determined by the generalized born model (GB/SA) (see, e.g., goshelke and Case, computational Chemistry, volume 25,Issue 2,Pages 238-250 (2004)). Beyond the common rigid receptor approximation in structure-based virtual screening, the induced fit docking scheme predicts the effect of ligand docking on protein structure.

Glide docking and scoring method

The Glide HTVS, SP and XP docking methods are well known. Glide HTVS and SP use a series of hierarchical filters to find possible positions of ligands in the region of the receptor binding site. The shape and nature of the receptors are represented on the grid by different sets of domains that provide progressively more accurate scoring of ligand positions. An exhaustive list of ligand torsions yields a collection of ligand conformations that are examined during docking. Given these ligand conformations, initial screening is performed deterministically across the entire phase space available for ligands to locate promising ligand positions. From the poses selected by the initial screening, the ligands were refined in the torsion space in the acceptor domain using OPLS34 (Glide SP & XP) or OPLS2005 (GLIDE HTVS) with distance-dependent dielectric models. Finally, in the acceptor domain with full ligand flexibility, a small number of poses can be minimized (post-docking minimization or PDM).

The molecular mechanical energy of the combined poisson-boltzmann or generalized born and surface area continuous solvation (MM/PBSA and MM/GBSA) method can be used to estimate the free energy of binding of small ligands to biological macromolecules (geniden S, ryde u.t MM/PBSAand MM/GBSAmethods to estimate ligand-binding affinis.

Lead compounds identified using these methods can be evaluated for their effectiveness as potential therapeutic agents by assessing their adsorption, distribution, metabolism and excretion (collectively referred to as ADME) as well as toxicity in vivo, in vitro, or in silico. One representative method of computer evaluation of adsorption, distribution, metabolism, and excretion (collectively referred to as ADME) is QikProp (Schrodinger). QiaProp predicted a broad range of predictive properties including octanol/water and water/gas log Ps, log S, log BB, total CNS activity, caco-2 and MDCK cell permeabilities, log Khsa of human serum albumin binding and log IC of HERGK+ channel blockage ₅₀ . This allows determining the suitability of the molecule as a potential therapeutic agent. The prediction of QikProp is based on the complete 3D molecular structure and thus can provide accurate results to predict the properties of molecules with novel scaffolds as well-known drug analogs. QikProp rapidly screens the compound library to obtain useful matching results, identifying molecules with calculated properties that are outside the normal range of known drugs, and thus easily screening candidate molecules with unsuitable ADME properties.

One representative method of electronic toxicity assessment is Derek Nexus. Derek Nexus predicts the potential toxicity of most toxicological endpoints including carcinogenicity, mutagenicity, genotoxicity, skin sensitization, teratogenicity, irritation, respiratory sensitization and reproductive toxicity.

Thus, the computer ability of compounds to bind to various receptors can be assessed using docking-Glide SP/XP MM-GBSA (or MM-PBSA can be used), docking with RORB/C & RORA inverse agonist structures. Using this approach, libraries of benzodiazepine molecules are screened for potential CNS activity. The following lead compounds were identified.

While not wishing to be bound by a particular theory, it is believed that substitution on the aromatic ring does not significantly alter the activity of the compound as a RORA agonist or a GABA-A agonist, or its ability to cross the blood brain barrier. Thus, compounds of the general formula are expected to exhibit binding affinity for RORA receptors, agonist activity after binding, selectivity for RORA over GABA-A and/or to be unable or less able to cross the blood brain barrier:

/>

/>

and pharmaceutically acceptable salts and prodrugs thereof, wherein R ² And u is as defined above for formula A, except that u may be 0 and n is 0, 1 or 2.

Based on the information obtained using the screening assays discussed in this example, it is believed that the compounds of formula B-H will be agonists of the RORA receptor, will bind to the RORA receptor with high affinity, will not bind to GABA receptors such as GABA-a receptors with high affinity, and will not cross the blood brain barrier. Using the screening assays described in this example, individual compounds can be tested to confirm these properties.

The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incorporated herein by reference in their entirety.

Claims (68)

1. A method for treating, preventing, reducing the susceptibility to, reducing the severity of, or slowing the progression of a disorder selected from the group consisting of pancreatitis, sarcopenia, stroke, glioblastoma, and traumatic brain injury, comprising administering to a patient in need thereof an effective amount of a compound of formula (a):

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

Wherein one of X and Z is selected from the group consisting of-NH-, -N (NH) ₂ )-、-N(OH)-、-N(CH ₂ -O-P(O)(OH) ₂ )-；N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -, -O-, -CH ₂ -、-CH(C _1-10 Alkyl) -, C (C) _1-10 Alkyl group ₂ -、-CH(C _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ )-、-CH(OH)-、-CH(O-C _1-10 Alkyl) -, CH (NH) ₂ )-、-CH(NH-C _1-10 Alkyl) -and-CH (C (O) NH ₂ ) -a group consisting of a group of,

and the other of X and Z is selected from the group consisting of-C (O) -, -SO ₂ -、-N(C(O)-、-CH ₂ -、-CH(C _1-10 Alkyl) -, C (C) _1-10 Alkyl group ₂ -、-CH(C _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ )-、-CH(NHC _1-10 Alkyl) -and-CH (C (O) NH ₂ ) -a group consisting of a group of,

y is selected from the group consisting of-NH, -N (NH) ₂ )-、-N(OH)-、-N(CH ₂ -O-P(O)(OH) ₂ )-；N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -, or-N (heteroaryl) -, -O-, -CH ₂ -、-CH(C _1-10 Alkyl) -, CH (C) _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -C (C) _1-10 Alkyl group ₂ -、-CF ₂ -、-CCl ₂ -、-CH(CF ₃ )-、-CH(OH)-、-CH(O-C _1-10 Alkyl) -, -C (O) -, -SO ₂ -、-N(C(O)-C _1-10 Alkyl) -, -N (C (O) O-C _1-10 Alkyl) -, CH (NH) ₂ )-、-CH(NH-C _1-10 Alkyl) -and-CH (C (O) NH ₂ ) -a group consisting of a group of,

a and B are independently phenyl, a five membered heteroaromatic ring containing one, two or three nitrogen, oxygen or sulfur atoms, or a six membered heteroaromatic ring containing one, two or three nitrogen atoms;

u and v are independently 0, 1, 2, 3 or 4; provided that at least one of u and v is 1, 2, 3 or 4;

each R ¹ And R is ² Independently R is ³ 、OH、OR ³ 、SR ³ 、S(O)R ³ 、SO ₂ R ³ 、C(O)R ³ 、C(O)OR ³ 、OC(O)R ³ 、OC(O)OR ³ 、NH ₂ 、NHR ³ 、NHC(O)R ³ 、NR ³ C(O)R ³ 、NHS(O) ₂ R ³ 、NR ³ S(O) ₂ R ³ 、NHC(O)OR ³ 、NR ³ C(O)OR ³ 、NHC(O)NH ₂ 、NHC(O)NHR ³ 、NHC(O)N(R ³ ) ₂ 、NR ³ C(O)N(R ³ ) ₂ 、C(O)NH ₂ 、C(O)NHR ³ 、C(O)N(R ³ ) ₂ 、C(O)NHOH、C(O)NHOR ³ 、C(O)NHSO ₂ R ³ 、C(O)NR ³ SO ₂ R ³ 、SO ₂ NH ₂ 、SO ₂ NHR ³ 、SO ₂ N(R ³ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ³ 、C(N)N(R ³ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), -CH ₂ Phosphonates, -CH ₂ O-phosphate, CH ₂ P(O)(OH) ₂ 、CH ₂ P(O)(OR ³ ) ₂ 、CH ₂ P(O)(OR ³ )(NR ³ )、CH ₂ P(O)(NR ³ ) ₂ 、CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) or CH ₂ A cyclopSal monophosphate prodrug,

wherein the term phosphate includes mono-, di-, tri-and stabilized phosphate prodrugs, the term phosphonate includes the same prodrugs present in phosphate prodrugs,

and when R is ¹ And R is ² On adjacent carbons, they may together form a saturated or unsaturated alkyl, aromatic or heteroaromatic ring,

each R ³ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁴ 、OH、OR ⁴ 、SR ⁴ 、S(O)R ⁴ 、SO ₂ R ⁴ 、C(O)R ⁴ 、C(O)OR ⁴ 、OC(O)R ⁴ 、OC(O)OR ⁴ 、NH ₂ 、NHR ⁴ 、NHC(O)R ⁴ 、NR ⁴ C(O)R ⁴ 、NHS(O) ₂ R ⁴ 、NR ⁴ S(O) ₂ R ⁴ 、NHC(O)OR ⁴ 、NR ⁴ C(O)OR ⁴ 、NHC(O)NH ₂ 、NHC(O)NHR ⁴ 、NHC(O)N(R ⁴ ) ₂ 、NR ⁴ C(O)N(R ⁴ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁴ 、C(O)N(R ⁴ ) ₂ 、C(O)NHOH、C(O)NHOR ⁴ 、C(O)NHSO ₂ R ⁴ 、C(O)NR ⁴ SO ₂ R ⁴ 、SO ₂ NH ₂ 、SO ₂ NHR ⁴ 、SO ₂ N(R ⁴ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁴ 、C(N)N(R ⁴ ) ₂ 、C(N)OH、C(N)OCH ⁴ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl), cyclophosphate prodrugs, CH ₂ P(O)(OH) ₂ 、CH ₂ P(O)(OR ⁴ ) ₂ 、CH ₂ P(O)(OR ⁴ )(NR ⁴ )、CH ₂ P(O)(NR ⁴ ) ₂ 、CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and CH ₂ A cyclopSal monophosphate prodrug,

each R ⁴ Independently selected from aryl, heteroaryl, arylalkyl, alkylaryl, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁵ 、OH、OR ⁵ 、SR ⁵ 、S(O)R ⁵ 、SO ₂ R ⁵ 、C(O)R ⁵ 、C(O)OR ⁵ 、OC(O)R ⁵ 、OC(O)OR ⁵ 、NH ₂ 、NHR ⁵ 、NHC(O)R ⁵ 、NR ⁵ C(O)R ⁵ 、NHS(O) ₂ R ⁵ 、NR ⁵ S(O) ₂ R ⁵ 、NHC(O)OR ⁵ 、NR ⁵ C(O)OR ⁵ 、NHC(O)NH ₂ 、NHC(O)NHR ⁵ 、NHC(O)N(R ⁵ ) ₂ 、NR ⁵ C(O)N(R ⁵ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁵ 、C(O)N(R ⁵ ) ₂ 、C(O)NHOH、C(O)NHOR ⁵ 、C(O)NHSO ₂ R ⁵ 、C(O)NR ⁵ SO ₂ R ⁵ 、SO ₂ NH ₂ 、SO ₂ NHR ⁵ 、SO ₂ N(R ⁵ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁵ 、C(N)N(R ⁵ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

each R ⁵ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁶ 、OH、OR ⁶ 、SR ⁶ 、S(O)R ⁶ 、SO ₂ R ⁶ 、C(O)R ⁶ 、C(O)OR ⁶ 、OC(O)R ⁶ 、OC(O)OR ⁶ 、NH ₂ 、NHR ⁶ 、NHC(O)R ⁶ 、NR ⁶ C(O)R ⁶ 、NHS(O) ₂ R ⁶ 、NR ⁶ S(O) ₂ R ⁶ 、NHC(O)OR ⁶ 、NR ⁶ C(O)OR ⁶ 、NHC(O)NH ₂ 、NHC(O)NHR ⁶ 、NHC(O)N(R ⁶ ) ₂ 、NR ⁶ C(O)N(R ⁶ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁶ 、C(O)N(R ⁶ ) ₂ 、C(O)NHOH、C(O)NHOR ⁶ 、C(O)NHSO ₂ R ⁶ 、C(O)NR ⁶ SO ₂ R ⁶ 、SO ₂ NH ₂ 、SO ₂ NHR ⁶ 、SO ₂ N(R ⁶ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁶ 、C(N)N(R ⁶ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ 、F、Cl、Br、I、P(O)(OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

each R ⁶ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: c (C) _1-10 Alkyl, C _2-10 Alkenyl, C _2-10 Alkynyl, OH, NH ₂ 、C(O)NH _2、 C(O)NHOH、SO ₂ NH _2、 COOH、C(O)H、C(N)NH ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

or a pharmaceutically acceptable salt or prodrug thereof.

2. The method of claim 1, wherein the compound is a retinoic acid receptor-like orphan receptor (ROR) alpha agonist.

3. The method of claim 1, wherein one of X and Z is-C (O) -, -SO ₂ -or-NC (O) -, and the other is-NH-, -N (NH) ₂ )-、-N(OH)-、-N(CH ₂ -O-P(O)(OH) ₂ )-；-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -or-O-.

4. The method of claim 1, wherein one of X and Z is-C (O) -, -SO ₂ -or-N (C (O) -, and the other is-CH ₂ -、-CH(C _1-6 Alkyl) -, C (alkyl) ₂ -、-CH(C _3-8 Cycloalkyl) -, CH (C) _2-6 Alkenyl, -CH (C) _2-6 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ ) -, -CH (NH alkyl) -or-CH (C (O) NH) ₂ )-。

5. The method of claim 1, wherein one of X and Z is-NH-, -N (CH) ₂ -O-P(O)(OH) ₂ )-；-N(NH ₂ ) -, -N (OH) -, -N (alkyl) -or-O-, and the other is-CH ₂ -、-CH(C _1-6 Alkyl) -, C (alkyl) ₂ -、-CH(C _3-8 Cycloalkyl) -, CH (C) _2-6 Alkenyl, -CH (C) _2-6 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ ) -, -CH (NH alkyl) -or-CH (C (O) NH) ₂ )-。

6. The method of claim 1, wherein one of X and Z is-NH-, -N (NH) ₂ )-、-N(CH ₂ -O-P(O)(OH) ₂ )-；-N(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -, and the other is-C (O) -or-SO ₂ -。

7. The method of claim 1, wherein Y is-NH, -N (NH) ₂ )-、-N(CH ₂ -O-P(O)(OH) ₂ )-；-NH(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -or-O-.

8. The method of claim 6, wherein Y is-NH, -N (NH) ₂ )-、-N(CH ₂ -O-P(O)(OH) ₂ )-；-N(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -,

9. the method of claim 1, wherein R ¹ And R is ² One is H, -CH ₂ Phosphonates, -CH ₂ O-phosphates, wherein the term phosphate includes mono-, di-, tri-and stabilized phosphate prodrugs, and the term phosphonate includes the same prodrugs present in phosphate prodrugs.

10. The method of claim 1, wherein R ¹ And R is ² One of them is H, -CH ₂ P(O)(OH) ₂ 、-CH ₂ P(O)(OH)(OR ⁶ )、-CH ₂ P(O)(OR ⁶ ) ₂ 、-CH ₂ P(O)(OR ⁶ )(NR ⁶ )、-CH ₂ P(O)(NR ⁶ ) ₂ 、-CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) or-CH ₂ -a cyclopsala monophosphate prodrug.

11. The method of claim 9, wherein R ¹ And R is ² One of them is a phosphonate, phosphoramidate, cyclopsala monophosphate prodrug, or of the formula-CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl).

12. The method of claim 1, wherein R ¹ And R is ² One of them is C (O) NHR ⁴ 、C(O)(NR ⁴ ) ₂ ，

Formed into-C (O) R ⁴ .

Wherein R is ⁴ Is C _1-10 Alkyl, C _3-10 Cycloalkyl, C _2-10 Alkenyl, C _2-10 Alkynyl, C _1-10 Haloalkyl, C _1-10 Alkyl-aryl or C _1-10 Haloalkyl-aryl, and m is 0, 1 or 2.

13. The method of claim 1, wherein R ¹ And R is ² One of them is-C (O) -C _1-10 Alkyl, -C (O) -alkylaryl, -C (O) -heterocyclyl-CH ₂ -aryl, -C (O) -heterocyclyl-CF ₂ -aryl, -C (O) -cycloalkyl-alkylaryl, -C (O) NHC _1-10 Alkyl, -C (O) NH-alkylaryl, -C (O) NH-heterocyclyl-CF ₂ -aryl, -C (O) NH-cycloalkyl-alkylaryl, -SO ₂ -C _1-10 Alkyl, -SO ₂ -alkylaryl, -SO ₂ -heterocyclyl-alkylaryl, -SO ₂ -heterocyclyl-CF ₂ -aryl or-SO _2- Cycloalkyl-alkylaryl.

14. The method of claim 1, wherein the compound has one of the following formulas:

or a pharmaceutically acceptable salt or prodrug thereof.

15. The method of claim 1, wherein the compound has the formula:

or a pharmaceutically acceptable salt or prodrug thereof.

16. The method of any one of claims 1 to 14, wherein the compound is administered in a composition, wherein the composition comprises a pharmaceutically acceptable carrier or excipient.

17. The method of claim 15, wherein the composition is a transdermal composition or a nanoparticle composition.

18. The method of any one of claims 1-14, further comprising administering a second retinoic acid receptor-like orphan receptor (ROR) modulator from formula (a).

19. The method of any one of claims 1-14, further comprising administering one or more additional active agents for treating pancreatitis, sarcopenia, stroke, or traumatic brain injury.

20. The method of claim 15, further comprising administering one or more active agents selected from the group consisting of agents for treating pancreatitis, sarcopenia, stroke, or traumatic brain injury.

21. The method according to any one of claims 1 to 14, wherein the pancreatitis is hypertriglyceridemia-induced pancreatitis, pancreatitis resulting from iatrogenic diseases (pancreatitis in view of ERCP surgery), pancreatitis resulting from gall stones, or pancreatitis resulting from drinking alcohol.

22. The method according to any one of claims 1 to 14, wherein the compound of formula (a) is administered before, simultaneously with or after a treatment and/or surgery associated with an increased risk of pancreatitis.

23. A pharmaceutical composition comprising a compound of any one of claims 1 to 14 and one or more active agents selected from the group consisting of statins, ACE inhibitors, oral contraceptives/Hormone Replacement Therapy (HRT), diuretics, antiretroviral therapy, valproic acid, oral hypoglycemic agents and combinations thereof.

24. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14 and one or more active agents selected from the group consisting of blood diluents, compounds that break down existing blood clots, platelet aggregation inhibitors, anticoagulants, neuroprotective agents, argatroban, alfimeprase, tenecteplase, ancrod, sildenafil, insulin growth factor, magnesium sulfate, human serum albumin, caffeine alcohol, microfibrlysin, statin, eptifibatide, tinzaparin, etanercept, citicoline, edaravone, cilostazol and combinations thereof.

25. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14 and one or more active agents selected from the group consisting of tranexamic acid, sedatives, analgesics, paralytic agents, antiepileptic drugs, norepinephrine, insulin and VLA-1 (very late activating antigen-I) antagonists.

26. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14 and one or more active agents selected from temozolomide, cannabinoids, berberine, perillyl alcohol, radiosensitizers, boron neutron capture agents, anticonvulsants, corticosteroids, chimeric Antigen Receptor (CAR) T cells using CLTX, IL13 ra 2, her2/CMV, EGFRvIII, CSPG4, NKG2DL, CD19 or CD133 as targeting domain, MP-Pt (IV), RIPGBM and (Ribociclib).

27. Use of a compound of formula a in the manufacture of a medicament for the treatment, prevention, reduction of susceptibility to, reduction of severity of, or delay of progression of a disorder selected from the group consisting of pancreatitis, sarcopenia, stroke, glioblastoma, and traumatic brain injury, wherein formula (a) has the structure:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

wherein one of X and Z is selected from the group consisting of-NH-, -N (NH) ₂ )-、-N(OH)-、-N(CH ₂ -O-P(O)(OH) ₂ )-；N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -, -O-, -CH ₂ -、-CH(C _1-10 Alkyl) -, C (C) _1-10 Alkyl group ₂ -、-CH(C _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ )-、-CH(OH)-、-CH(O-C _1-10 Alkyl) -, CH (NH) ₂ )-、-CH(NH-C _1-10 Alkyl) -and-CH (C (O) NH ₂ ) -a group consisting of a group of,

and the other of X and ZSelected from the group consisting of-C (O) -, -SO ₂ -、-N(C(O)-、-CH ₂ -、-CH(C _1-10 Alkyl) -, C (C) _1-10 Alkyl group ₂ -、-CH(C _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ )-、-CH(NHC _1-10 Alkyl) -and-CH (C (O) NH ₂ ) -a group consisting of a group of,

y is selected from the group consisting of-NH, -N (NH) ₂ )-、-N(OH)-、-N(CH ₂ -O-P(O)(OH) ₂ )-；N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -, or-N (heteroaryl) -, -O-, -CH ₂ -、-CH(C _1-10 Alkyl) -, CH (C) _3-10 Cycloalkyl) -, CH (C) _2-10 Alkenyl, -CH (C) _2-10 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -C (C) _1-10 Alkyl group ₂ -、-CF ₂ -、-CCl ₂ -、-CH(CF ₃ )-、-CH(OH)-、-CH(O-C _1-10 Alkyl) -, -C (O) -, -SO2-, -N (C (O) -C _1-10 Alkyl) -, -N (C (O) O-C _1-10 Alkyl) -, CH (NH) ₂ )-、-CH(NH-C _1-10 Alkyl) -and-CH (C (O) NH ₂ ) -a group consisting of a group of,

a and B are independently phenyl, a five membered heteroaromatic ring containing one, two or three nitrogen, oxygen or sulfur atoms, or a six membered heteroaromatic ring containing one, two or three nitrogen atoms;

u and v are independently 0, 1, 2, 3 or 4; provided that at least one of u and v is 1, 2, 3 or 4;

each R ¹ And R is ² Independently R is ³ 、OH、OR ³ 、SR ³ 、S(O)R ³ 、SO ₂ R ³ 、C(O)R ³ 、C(O)OR ³ 、OC(O)R ³ 、OC(O)OR ³ 、NH ₂ 、NHR ³ 、NHC(O)R ³ 、NR ³ C(O)R ³ 、NHS(O) ₂ R ³ 、NR ³ S(O) ₂ R ³ 、NHC(O)OR ³ 、NR ³ C(O)OR ³ 、NHC(O)NH ₂ 、NHC(O)NHR ³ 、NHC(O)N(R ³ ) ₂ 、NR ³ C(O)N(R ³ ) ₂ 、C(O)NH ₂ 、C(O)NHR ³ 、C(O)N(R ³ ) ₂ 、C(O)NHOH、C(O)NHOR ³ 、C(O)NHSO ₂ R ³ 、C(O)NR ³ SO ₂ R ³ 、SO ₂ NH ₂ 、SO ₂ NHR ³ 、SO ₂ N(R ³ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ³ 、C(N)N(R ³ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), -CH ₂ Phosphonates, -CH ₂ O-phosphate, CH ₂ P(O)(OH) ₂ 、CH ₂ P(O)(OR ³ ) ₂ 、CH ₂ P(O)(OR ³ )(NR ³ )、CH ₂ P(O)(NR ³ ) ₂ 、CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) or CH ₂ A cyclopSal monophosphate prodrug,

wherein the term phosphate includes mono-, di-, tri-and stabilized phosphate prodrugs, the term phosphonate includes the same prodrugs present in phosphate prodrugs,

and when R is ¹ And R is ² On adjacent carbons, they may together form a saturated or unsaturated alkyl, aromatic or heteroaromatic ring,

each R ³ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁴ 、OH、OR ⁴ 、SR ⁴ 、S(O)R ⁴ 、SO ₂ R ⁴ 、C(O)R ⁴ 、C(O)OR ⁴ 、OC(O)R ⁴ 、OC(O)OR ⁴ 、NH ₂ 、NHR ⁴ 、NHC(O)R ⁴ 、NR ⁴ C(O)R ⁴ 、NHS(O) ₂ R ⁴ 、NR ⁴ S(O) ₂ R ⁴ 、NHC(O)OR ⁴ 、NR ⁴ C(O)OR ⁴ 、NHC(O)NH ₂ 、NHC(O)NHR ⁴ 、NHC(O)N(R ⁴ ) ₂ 、NR ⁴ C(O)N(R ⁴ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁴ 、C(O)N(R ⁴ ) ₂ 、C(O)NHOH、C(O)NHOR ⁴ 、C(O)NHSO ₂ R ⁴ 、C(O)NR ⁴ SO ₂ R ⁴ 、SO ₂ NH ₂ 、SO ₂ NHR ⁴ 、SO ₂ N(R ⁴ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁴ 、C(N)N(R ⁴ ) ₂ 、C(N)OH、C(N)OCH ⁴ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl), cyclophosphate prodrugs, CH ₂ P(O)(OH) ₂ 、CH ₂ P(O)(OR ⁴ ) ₂ 、CH ₂ P(O)(OR ⁴ )(NR ⁴ )、CH ₂ P(O)(NR ⁴ ) ₂ 、CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and CH ₂ A cyclopSal monophosphate prodrug,

each R ⁴ Independently selected from aryl, heteroaryl, arylalkyl, alkylaryl, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁵ 、OH、OR ⁵ 、SR ⁵ 、S(O)R ⁵ 、SO ₂ R ⁵ 、C(O)R ⁵ 、C(O)OR ⁵ 、OC(O)R ⁵ 、OC(O)OR ⁵ 、NH ₂ 、NHR ⁵ 、NHC(O)R ⁵ 、NR ⁵ C(O)R ⁵ 、NHS(O) ₂ R ⁵ 、NR ⁵ S(O) ₂ R ⁵ 、NHC(O)OR ⁵ 、NR ⁵ C(O)OR ⁵ 、NHC(O)NH ₂ 、NHC(O)NHR ⁵ 、NHC(O)N(R ⁵ ) ₂ 、NR ⁵ C(O)N(R ⁵ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁵ 、C(O)N(R ⁵ ) ₂ 、C(O)NHOH、C(O)NHOR ⁵ 、C(O)NHSO ₂ R ⁵ 、C(O)NR ⁵ SO ₂ R ⁵ 、SO ₂ NH ₂ 、SO ₂ NHR ⁵ 、SO ₂ N(R ⁵ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁵ 、C(N)N(R ⁵ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

each R ⁵ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁶ 、OH、OR ⁶ 、SR ⁶ 、S(O)R ⁶ 、SO ₂ R ⁶ 、C(O)R ⁶ 、C(O)OR ⁶ 、OC(O)R ⁶ 、OC(O)OR ⁶ 、NH ₂ 、NHR ⁶ 、NHC(O)R ⁶ 、NR ⁶ C(O)R ⁶ 、NHS(O) ₂ R ⁶ 、NR ⁶ S(O) ₂ R ⁶ 、NHC(O)OR ⁶ 、NR ⁶ C(O)OR ⁶ 、NHC(O)NH ₂ 、NHC(O)NHR ⁶ 、NHC(O)N(R ⁶ ) ₂ 、NR ⁶ C(O)N(R ⁶ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁶ 、C(O)N(R ⁶ ) ₂ 、C(O)NHOH、C(O)NHOR ⁶ 、C(O)NHSO ₂ R ⁶ 、C(O)NR ⁶ SO ₂ R ⁶ 、SO ₂ NH ₂ 、SO ₂ NHR ⁶ 、SO ₂ N(R ⁶ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁶ 、C(N)N(R ⁶ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ 、F、Cl、Br、I、P(O)(OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

each R ⁶ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: c (C) _1-10 Alkyl, C _2-10 Alkenyl, C _2-10 Alkynyl, OH, NH ₂ 、C(O)NH _2、 C(O)NHOH、SO ₂ NH _2、 COOH、C(O)H、C(N)NH ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

or a pharmaceutically acceptable salt or prodrug thereof.

28. The use of claim 26, wherein the compound is a retinoic acid receptor-like orphan receptor (ROR) alpha agonist.

29. The use according to claim 26, wherein one of X and Z is-C (O) -, -SO ₂ -or-NC (O) -, and the other is-NH-, -N (NH) ₂ )-、-N(OH)-、-N(CH ₂ -O-P(O)(OH) ₂ )-；-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -, or-O-.

30. The use according to claim 26, wherein one of X and Z is-C (O) -, -SO ₂ -or-N (C (O) -, and the other is-CH ₂ -、-CH(C _1-6 Alkyl) -, C (alkyl) ₂ -、-CH(C _3-8 Cycloalkyl) -, CH (C) _2-6 Alkenyl, -CH (C) _2-6 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ ) -, -CH (NH alkyl) -or-CH (C (O) NH) ₂ )-。

31. The use according to claim 26, wherein one of X and Z is-NH-, -N (CH) ₂ -O-P(O)(OH) ₂ )-；-N(NH ₂ ) -, -N (OH) -, -N (alkyl) -or-O-, and the other is-CH ₂ -、-CH(C _1-6 Alkyl) -, C (alkyl) ₂ -、-CH(C _3-8 Cycloalkyl) -, CH (C) _2-6 Alkenyl, -CH (C) _2-6 Alkynyl) -, -CH (aryl) -, -CH (heteroaryl) -, -CF ₂ -、-CCl ₂ -、-CH(CF ₃ ) -, -CH (OH) -, -CH (Oalkyl) -, -CH (NH) ₂ ) -, -CH (NH alkyl) -or-CH (C (O) NH) ₂ )-。

32. The use according to claim 26, wherein one of X and Z is-NH-, -N (NH) ₂ )-、-N(CH ₂ -O-P(O)(OH) ₂ )-；-N(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -, and anotherEach is-C (O) -or-SO ₂ -。

33. The use according to claim 26, wherein Y is-NH, -N (NH) ₂ )-、-N(CH ₂ -O-P(O)(OH) ₂ )-；-NH(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -or-O-.

34. The use according to claim 26, wherein Y is-NH, -N (NH) ₂ )-、-N(CH ₂ -O-P(O)(OH) ₂ )-；-N(OH)-、-N(C _1-10 Alkyl) -, N (C) _3-10 Cycloalkyl) -, N (C) _2-10 Alkenyl) -, N (C) _2-10 Alkynyl) -, -N (aryl) -or-N (heteroaryl) -.

35. The use according to claim 26, wherein R ¹ And R is ² One is H, -CH ₂ Phosphonates, -CH ₂ O-phosphates, wherein the term phosphate includes mono-, di-, tri-and stabilized phosphate prodrugs, and the term phosphonate includes the same prodrugs present in phosphate prodrugs.

36. The use according to claim 26, wherein R ¹ And R is ² One of them is H, -CH ₂ P(O)(OH) ₂ 、-CH ₂ P(O)(OH)(OR ⁶ )、-CH ₂ P(O)(OR ⁶ ) ₂ 、-CH ₂ P(O)(OR ⁶ )(NR ⁶ )、-CH ₂ P(O)(NR ⁶ ) ₂ 、-CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) or-CH ₂ -a cyclopsala monophosphate prodrug.

37. The use according to claim 35, wherein R ¹ And R is ² One of them is a phosphonate, phosphoramidate, cyclopsala monophosphate prodrug, or of the general formula-CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl).

38. The use according to claim 26, wherein R ¹ And R is ² One of them is C (O) NHR ⁴ 、C(O)(NR ⁴ ) ₂ 、

or-C (O) R ⁴ .

Wherein R is ⁴ Is C _1-10 Alkyl, C _3-10 Cycloalkyl, C _2-10 Alkenyl, C _2-10 Alkynyl, C _1-10 Haloalkyl, C _1-10 Alkyl-aryl or C _1-10 Haloalkyl-aryl, and m is 0, 1 or 2.

39. The use according to claim 26, wherein R ¹ And R is ² One of them is-C (O) -C _1-10 Alkyl, -C (O) -alkylaryl, -C (O) -heterocyclyl-CH ₂ -aryl, -C (O) -heterocyclyl-CF ₂ -aryl, -C (O) -cycloalkyl-alkylaryl, -C (O) NHC _1-10 Alkyl, -C (O) NH-alkylaryl, -C (O) NH-heterocyclyl-CF ₂ -aryl, -C (O) NH-cycloalkyl-alkylaryl, -SO ₂ -C _1-10 Alkyl, -SO ₂ -alkylaryl, -SO ₂ -heterocyclyl-alkylaryl, -SO ₂ -heterocyclyl-CF ₂ -aryl or-SO _2- Cycloalkyl-alkylaryl.

40. The use of claim 26, wherein the compound has one of the following formulas:

/>

or a pharmaceutically acceptable salt or prodrug thereof.

41. The use of claim 26, wherein the compound has the general formula:

Or a pharmaceutically acceptable salt or prodrug thereof.

42. The use of any one of claims 26 to 40, wherein the compound is administered in a composition, wherein the composition comprises a pharmaceutically acceptable carrier or excipient.

43. The use according to claim 41, wherein the composition is a transdermal composition or a nanoparticle composition.

44. The use of any one of claims 26 to 40, further comprising administering a second retinoic acid receptor-like orphan receptor (ROR) modulator from formula (a).

45. The use of any one of claims 26 to 40, further comprising administering one or more additional active agents for treating pancreatitis, sarcopenia, stroke, or traumatic brain injury.

46. The use according to claim 41, further comprising administering one or more active agents selected from the group consisting of agents for treating pancreatitis, sarcopenia, stroke, or traumatic brain injury.

47. The use according to any one of claims 26 to 40, wherein the pancreatitis is hypertriglyceridemia-induced pancreatitis, pancreatitis resulting from iatrogenic diseases (pancreatitis in view of ERCP surgery), pancreatitis resulting from gall stones, or pancreatitis resulting from drinking alcohol.

48. The method of any one of claims 26 to 40, wherein the compound of formula (a) is administered prior to, simultaneously with, or after treatment and/or surgery associated with increased risk of pancreatitis.

49. A method for treating, preventing, reducing the susceptibility to, reducing the severity of, or slowing the progression of a disorder selected from the group consisting of pancreatitis, sarcopenia, stroke, glioblastoma, and traumatic brain injury, comprising administering to a patient in need thereof an effective amount of a compound of formulae (B) - (H):

/>

/>

u is independently 0, 1, 2, 3 or 4;

n is independently 0, 1 or 2,

each R ² Independently R is ³ 、OH、OR ³ 、SR ³ 、S(O)R ³ 、SO ₂ R ³ 、C(O)R ³ 、C(O)OR ³ 、OC(O)R ³ 、OC(O)OR ³ 、NH ₂ 、NHR ³ 、NHC(O)R ³ 、NR ³ C(O)R ³ 、NHS(O) ₂ R ³ 、NR ³ S(O) ₂ R ³ 、NHC(O)OR ³ 、NR ³ C(O)OR ³ 、NHC(O)NH ₂ 、NHC(O)NHR ³ 、NHC(O)N(R ³ ) ₂ 、NR ³ C(O)N(R ³ ) ₂ 、C(O)NH ₂ 、C(O)NHR ³ 、C(O)N(R ³ ) ₂ 、C(O)NHOH、C(O)NHOR ³ 、C(O)NHSO ₂ R ³ 、C(O)NR ³ SO ₂ R ³ 、SO ₂ NH ₂ 、SO ₂ NHR ³ 、SO ₂ N(R ³ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ³ 、C(N)N(R ³ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), -CH ₂ Phosphonates, -CH ₂ O-phosphate, CH ₂ P(O)(OH) ₂ 、CH ₂ P(O)(OR ³ ) ₂ 、CH ₂ P(O)(OR ³ )(NR ³ )、CH ₂ P(O)(NR ³ ) ₂ 、CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) or CH ₂ A cyclopSal monophosphate prodrug,

wherein the term phosphate includes mono-, di-, tri-and stabilized phosphate prodrugs, the term phosphonate includes the same prodrugs present in phosphate prodrugs,

each R ³ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁴ 、OH、OR ⁴ 、SR ⁴ 、S(O)R ⁴ 、SO ₂ R ⁴ 、C(O)R ⁴ 、C(O)OR ⁴ 、OC(O)R ⁴ 、OC(O)OR ⁴ 、NH ₂ 、NHR ⁴ 、NHC(O)R ⁴ 、NR ⁴ C(O)R ⁴ 、NHS(O) ₂ R ⁴ 、NR ⁴ S(O) ₂ R ⁴ 、NHC(O)OR ⁴ 、NR ⁴ C(O)OR ⁴ 、NHC(O)NH ₂ 、NHC(O)NHR ⁴ 、NHC(O)N(R ⁴ ) ₂ 、NR ⁴ C(O)N(R ⁴ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁴ 、C(O)N(R ⁴ ) ₂ 、C(O)NHOH、C(O)NHOR ⁴ 、C(O)NHSO ₂ R ⁴ 、C(O)NR ⁴ SO ₂ R ⁴ 、SO ₂ NH ₂ 、SO ₂ NHR ⁴ 、SO ₂ N(R ⁴ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁴ 、C(N)N(R ⁴ ) ₂ 、C(N)OH、C(N)OCH ⁴ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl), cyclophosphate prodrugs, CH ₂ P(O)(OH) ₂ 、CH ₂ P(O)(OR ⁴ ) ₂ 、CH ₂ P(O)(OR ⁴ )(NR ⁴ )、CH ₂ P(O)(NR ⁴ ) ₂ 、CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and CH ₂ A cyclopSal monophosphate prodrug,

each R ⁴ Independently selected from aryl, heteroaryl, arylalkyl, alkylaryl, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁵ 、OH、OR ⁵ 、SR ⁵ 、S(O)R ⁵ 、SO ₂ R ⁵ 、C(O)R ⁵ 、C(O)OR ⁵ 、OC(O)R ⁵ 、OC(O)OR ⁵ 、NH ₂ 、NHR ⁵ 、NHC(O)R ⁵ 、NR ⁵ C(O)R ⁵ 、NHS(O) ₂ R ⁵ 、NR ⁵ S(O) ₂ R ⁵ 、NHC(O)OR ⁵ 、NR ⁵ C(O)OR ⁵ 、NHC(O)NH ₂ 、NHC(O)NHR ⁵ 、NHC(O)N(R ⁵ ) ₂ 、NR ⁵ C(O)N(R ⁵ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁵ 、C(O)N(R ⁵ ) ₂ 、C(O)NHOH、C(O)NHOR ⁵ 、C(O)NHSO ₂ R ⁵ 、C(O)NR ⁵ SO ₂ R ⁵ 、SO ₂ NH ₂ 、SO ₂ NHR ⁵ 、SO ₂ N(R ⁵ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁵ 、C(N)N(R ⁵ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

each R ⁵ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: r is R ⁶ 、OH、OR ⁶ 、SR ⁶ 、S(O)R ⁶ 、SO ₂ R ⁶ 、C(O)R ⁶ 、C(O)OR ⁶ 、OC(O)R ⁶ 、OC(O)OR ⁶ 、NH ₂ 、NHR ⁶ 、NHC(O)R ⁶ 、NR ⁶ C(O)R ⁶ 、NHS(O) ₂ R ⁶ 、NR ⁶ S(O) ₂ R ⁶ 、NHC(O)OR ⁶ 、NR ⁶ C(O)OR ⁶ 、NHC(O)NH ₂ 、NHC(O)NHR ⁶ 、NHC(O)N(R ⁶ ) ₂ 、NR ⁶ C(O)N(R ⁶ ) ₂ 、C(O)NH ₂ 、C(O)NHR ⁶ 、C(O)N(R ⁶ ) ₂ 、C(O)NHOH、C(O)NHOR ⁶ 、C(O)NHSO ₂ R ⁶ 、C(O)NR ⁶ SO ₂ R ⁶ 、SO ₂ NH ₂ 、SO ₂ NHR ⁶ 、SO ₂ N(R ⁶ ) ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)NHR ⁶ 、C(N)N(R ⁶ ) ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ 、F、Cl、Br、I、P(O)(OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

each R ⁶ Independently an aryl group, a heteroaryl group, C _3-10 Cycloalkyl, C _3-10 Cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, C _1-10 Alkyl, C _2-10 Alkenyl or C _2-10 Alkynyl, each of which is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: c (C) _1-10 Alkyl, C _2-10 Alkenyl, C _2-10 Alkynyl, OH, NH ₂ 、C(O)NH ₂ 、C(O)NHOH、SO ₂ NH ₂ 、COOH、C(O)H、C(N)NH ₂ 、C(N)OH、C(N)OCH ₃ 、CN、N ₃ 、NO ₂ 、CF ₃ 、CF ₂ CF ₃ 、OCF ₃ 、OCF ₂ CF ₃ Halogen (F, cl, br or I), P (O) (OH) ₂ 、P(O)(OR ⁴ ) ₂ 、P(O)(OR ⁴ )(NR ⁴ )、P(O)(NR ⁴ ) ₂ 、P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) and a cyclophosphate prodrug,

or a pharmaceutically acceptable salt or prodrug thereof.

50. The method of claim 48, wherein the compound is a retinoic acid receptor-like orphan receptor (ROR) alpha agonist.

51. The method of claim 48, wherein R is ¹ One is H, -CH ₂ Phosphonates, -CH ₂ O-phosphates, wherein the term phosphate includes mono-, di-, tri-and stabilized phosphate prodrugs, and the term phosphonate includes the same prodrugs present in phosphate prodrugs.

52. The method of claim 48, wherein R is ¹ One of them is H, -CH ₂ P(O)(OH) ₂ 、-CH ₂ P(O)(OH)(OR ⁶ )、-CH ₂ P(O)(OR ⁶ ) ₂ 、-CH ₂ P(O)(OR ⁶ )(NR ⁶ )、-CH ₂ P(O)(NR ⁶ ) ₂ 、-CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl) or-CH ₂ -a cyclopsala monophosphate prodrug.

53. The method of claim 51, wherein R is ¹ And R is ² One of them is a phosphonate, phosphoramidate, cyclopsala monophosphate prodrug, or of the formula-CH ₂ P(O)(OH)(OC _1-10 alkyl-O-C _1-20 Alkyl).

54. The method of claim 48, wherein R is ¹ One of them is C (O) NHR ⁴ 、C(O)(NR ⁴ ) ₂ 、

or-C (O) R ⁴ .

Wherein R is ⁴ Is C _1-10 Alkyl, C _3-10 Cycloalkyl, C _2-10 Alkenyl, C _2-10 Alkynyl, C _1-10 Haloalkyl, C _1-10 Alkyl-aryl or C _1-10 Haloalkyl-aryl and m is 0, 1 or 2.

55. The method of claim 48, wherein R is ¹ One of them is-C (O) -C _1-10 Alkyl, -C (O) -alkylaryl, -C (O) -heterocyclyl-CH ₂ -aryl, -C (O) -heterocyclyl-CF ₂ -aryl, -C (O) -cycloalkyl-alkylaryl, -C (O) NHC _1-10 Alkyl, -C (O) NH-alkylaryl, -C (O) NH-heterocyclyl-CF ₂ -aryl, -C (O) NH-cycloalkyl-alkylaryl, -SO ₂ -C _1-10 Alkyl, -SO ₂ -alkylaryl, -SO ₂ -heterocyclyl-alkylaryl, -SO ₂ -heterocyclyl-CF ₂ -aryl or-SO _2- Cycloalkyl-alkylaryl.

56. The method of claim 48, wherein the compound has the formula:

or a pharmaceutically acceptable salt or prodrug thereof.

57. The method of any one of claims 48 to 55, wherein the compound is administered in a composition, wherein the composition comprises a pharmaceutically acceptable carrier or excipient.

58. The method of claim 56, wherein the composition is a transdermal composition or a nanoparticle composition.

59. The method of any one of claims 48-55, further comprising administering a second retinoic acid receptor-like orphan receptor (ROR) modulator from formula (a).

60. The method of any one of claims 48 to 55, further comprising administering one or more additional active agents for treating pancreatitis, sarcopenia, stroke, or traumatic brain injury.

61. The method of claim 59, further comprising administering one or more active agents selected from the group consisting of agents for treating pancreatitis, sarcopenia, stroke, or traumatic brain injury.

62. The method of any one of claims 48 to 55, wherein the pancreatitis is hypertriglyceridemia-induced pancreatitis, pancreatitis resulting from iatrogenic disease (pancreatitis in view of ERCP surgery), pancreatitis resulting from gall stones, or pancreatitis resulting from drinking alcohol.

63. The method of any one of claims 48 to 55, wherein the compound of formulae (B) - (H) is administered prior to, simultaneously with, or after treatment and/or surgery associated with increased risk of pancreatitis.

64. A pharmaceutical composition comprising a compound according to any one of claims 48 to 55 and one or more active agents selected from the group consisting of statins, ACE inhibitors, oral contraceptives/Hormone Replacement Therapy (HRT), diuretics, antiretroviral therapy, valproic acid, oral hypoglycemic agents and combinations thereof.

65. A pharmaceutical composition comprising a compound of any one of claims 48 to 55 and one or more active agents selected from the group consisting of blood diluents, compounds that break down existing blood clots, platelet aggregation inhibitors, anticoagulants, neuroprotective agents, argatroban, alfimeprase, tenecteplase, ancrod, sildenafil, insulin growth factor, magnesium sulfate, human serum albumin, caffeine alcohol, microfibrlysin, statin, eptifibatide, tinzaparin, etanercept, citicoline, edaravone, cilostazol, and combinations thereof.

66. A pharmaceutical composition comprising a compound according to any one of claims 48 to 55 and one or more active agents selected from the group consisting of tranexamic acid, sedatives, analgesics, paralytic agents, antiepileptics, norepinephrine, insulin and VLA-1 (very late activating antigen-I) antagonists.

67. A pharmaceutical composition comprising a compound according to any one of claims 48 to 55 and one or more active agents selected from temozolomide, cannabinoids, berberine, perillyl alcohol, radiosensitizers, boron neutron capture agents, anticonvulsants, corticosteroids, chimeric Antigen Receptor (CAR) T cells using CLTX, IL13 ra 2, her2/CMV, EGFRvIII, CSPG4, NKG2DL, CD19 or CD133 as targeting domain, MP-Pt (IV), RIPGBM and (Ribociclib).

68. Use of a compound of formula (B) - (H) in the manufacture of a medicament for the treatment, prevention, reduction of susceptibility, reduction of severity or delay of progression of a disorder selected from the group consisting of pancreatitis, sarcopenia, stroke, glioblastoma, and traumatic brain injury.